EP3197907B1 - Vaccines against streptococcus pneumoniae serotype 8 - Google Patents

Vaccines against streptococcus pneumoniae serotype 8 Download PDF

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EP3197907B1
EP3197907B1 EP15770548.4A EP15770548A EP3197907B1 EP 3197907 B1 EP3197907 B1 EP 3197907B1 EP 15770548 A EP15770548 A EP 15770548A EP 3197907 B1 EP3197907 B1 EP 3197907B1
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glucopyranosyl
galactopyranosyl
saccharide
acid
amino
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EP3197907A1 (en
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Peter Seeberger
Claney Lebev PEREIRA
Chakkumkal ANISH
Benjamin SCHUMANN
Sharavathi Guddehalli PARAMESWARAPPA
Heung Sik Hahm
Subramanian GOVINDAN
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
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    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
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    • A61K39/085—Staphylococcus
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
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    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
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    • A61P27/16—Otologicals
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
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    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/18—Acyclic radicals, substituted by carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1271—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Micrococcaceae (F), e.g. Staphylococcus
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56944—Streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/315—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci
    • G01N2333/3156—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)

Definitions

  • the present invention relates to synthetic saccharides of general formula ( I ) that are related to Streptococcus pneumoniae serotype 8 capsular polysaccharide, conjugates thereof and the use of said saccharides and conjugates for raising a protective immune response in a human and/or animal host. Furthermore, the synthetic saccharide structures of general formula ( I ) are useful as marker in immunological assays for detection of antibodies against Streptococcus pneumoniae bacteria.
  • Streptococcus pneumoniae is a Gram-positive, encapsulated bacterium that is a main cause of infections of the respiratory tract and can lead to severe invasive pneumococcal disease (IPD). More than 90 different pneumococcal serotypes have been described to date. These are classified by the structure of their capsular polysaccharide (CPS), which is unique to each serotype. Consequently, the immune response generated against the CPS varies between different serotypes. This is used to generate specific antibodies in rabbits against the antigen of each serotype. Cross-reactivity between these specific antibodies and other serotypes than those they were raised against is often observed, due to structural similarities of the CPS of different serotypes. Due to its immunological properties, CPS is used as the main component of S. pneumoniae vaccines.
  • CPS capsular polysaccharide
  • the first efficient vaccine that contained the CPS of four different serotypes was described in 1945. It then took over thirty years until a vaccine was introduced that covered 14 serotypes, shortly followed by a 23-valent vaccine.
  • these polysaccharide vaccines had several shortcomings. They were not able to elicit a long-lasting protection and were not effective in the populations most vulnerable to infection, namely children under two years of age as well as immunodeficient and elderly patients. These shortcomings result from the immunology of carbohydrates and were overcome by the introduction of carbohydrate-protein conjugate vaccines.
  • the first pneumococcal conjugate vaccines were the seven-valent (PCV-7) and 10-valent (PCV-10) vaccine. PCV-7 was later replaced with the most recent vaccine (PCV-13), which contains the CPS-glycoconjugates of 13 different serotypes.
  • the currently marketed vaccines are effective in North America and Europe for individuals of a particular age.
  • the manufacturing process for these vaccines is complex and results in a higher price. Therefore, the vaccine is unaffordable in most developing countries. It is the object of the present invention to provide affordable synthetic saccharide vaccines that contain one of the most prevalent serotypes of the developing world.
  • Streptococcus pneumoniae serotype 8 has been associated with serotype replacement and cases of invasive pneumococcal disease increased steadily in the last decades.
  • Streptococcus pneumoniae type 8 is one of the prevalent S. pneumoniae serotypes.
  • the structure of the native Sp8 capsular polysaccharide repeating unit is a tetrasaccharide with the sequence ⁇ 4)-Glc ⁇ -(1 ⁇ 4)-Gal ⁇ -(1 ⁇ 4)-GlcA ⁇ -(1 ⁇ 4)-Glc ⁇ (1 ⁇ ( J. Am. Chem. Soc. 1957, 79 (11), 2787 ):
  • WO 9640225 A1 provides a Streptococcus pneumoniae serotype 8 oligosaccharide protein conjugate consisting of mixtures of 2 to 4 repeating units coupled using EDC to tetanus toxoid.
  • the mixtures of 2 to 4 repeating units are obtained by non-selective cleavage of the capsular polysaccharide followed by size exclusion chromatography. It is well known that oligosaccharides obtained by degradation of capsular polysaccharides present a high degree of heterogeneity due to co-isolated impurities and new epitopes that are introduced during degradation or as part of an immune evasion strategy by the pathogen.
  • oligosaccharides isolated from capsular polysaccharides comprise a set of potentially immunogenic epitopes depending on structural determinants that are recognized as "non-self" by the host immune system.
  • a drawback of the immunization with vaccines based on conjugates of isolated oligosaccharides, such as the conjugates of WO 9640225 A1 is the generation of a polyclonal immune response that is directed towards multiple immunogenic epitopes, among which some are non-protective (i.e. the antibodies that are elicited do not protect from diseases associated with S. pneumoniae type 8) or are immunodominant.
  • Such polyclonal immune response leads to toxicities that are not acceptable for a vaccine that is prophylactically administered to healthy (often infant) populations.
  • This drawback is overcome by the pure saccharide of the present invention, which presents a well-defined structure, a degree of purity suitable for clinical applications and no batch-to-batch variability.
  • WO 9640225 A1 discloses that a mixture of oligosaccharides containing 1 repeating unit i.e. a mixture of tetrasaccharides do not contain an immunogenic epitope and that at least mixtures of octasaccharides are required for preparing a conjugate against S. pneumoniae type 8.
  • the inventors have found that even short saccharides described herein contain a protective glycan epitope and are able to induce a protective immune response against S. pneumoniae serotype 8 bacteria in a human and/or animal host. Further references relating to S. pneumoniae can be found in WO2009/00826 A1 , WO2013/178236 A1 , Yano M.
  • a pure synthetic saccharide of general formula ( I ) that is related to the capsular polysaccharide of Streptococcus pneumoniae serotype 8 and contains a protective immunogenic glycan epitope i.e. a glycan epitope that is elicits and is recognized by antibodies which protect against diseases caused by S. pneumoniae type 8.
  • Said saccharide is suitable to be conjugated to an immunogenic carrier to provide a conjugate and a pharmaceutical composition thereof that are useful for prevention and/or treatment of diseases associated with Streptococcus pneumoniae, and more specifically against diseases associated with Streptococcus pneumoniae serotype 8.
  • the synthetic saccharide of general formula ( I ) is useful as marker in immunological assays for detection of antibodies against Streptococcus pneumoniae bacteria.
  • linker encompasses molecular fragments capable of connecting the reducing-end monosaccharide of a saccharide with an immunogenic carrier or a solid support, optionally by binding to at least one interconnecting molecule.
  • the function of the linker per se or together with the interconnecting molecule is to establish, keep and/or bridge a special distance between the reducing-end monosaccharide and an immunogenic carrier or a solid support. More specifically, one extremity of the linker is connected to the exocyclic oxygen atom at the anomeric center of the reducing-end monosaccharide and the other extremity is connected via the nitrogen atom with the interconnecting molecule, or directly with the immunogenic carrier or the solid support.
  • interconnecting molecule refers to a bifunctional molecule containing functional group X and functional group Y, wherein functional group X is capable of reacting with the terminal amino group on the linker L and the functional group Y is capable of reacting with a functionality present on an immunogenic carrier or on a solid support.
  • Figure 1 displays examples of commercially available interconnecting molecules, but does not restrict the interconnecting molecules that can be used according to the present invention to the examples displayed herein.
  • adjuvant refers to an immunological adjuvant i.e. a material used in a vaccine composition that modifies or augments the effects of said vaccine by enhancing the immune response to a given antigen contained in the vaccine without being antigenically related to it.
  • immunological adjuvant i.e. a material used in a vaccine composition that modifies or augments the effects of said vaccine by enhancing the immune response to a given antigen contained in the vaccine without being antigenically related to it.
  • adjuvants include:
  • each molecule or substance that is able to favor or amplify a particular situation in the cascade of immunological events, ultimately leading to a more pronounced immunological response can be defined as an adjuvant.
  • Saccharides are known by the person skilled in the art as TI-2 (T cell independent-2) antigens and poor immunogens. Therefore, to produce a saccharide-based vaccine, said saccharides are conjugated to an immunogenic carrier to provide a conjugate, which presents an increased immunogenicity in comparison with the saccharide.
  • immunogenic carrier is defined as a structure, which is conjugated to the saccharide to form a conjugate that presents an increased immunogenicity in comparison with the saccharide per se.
  • the conjugation of the saccharides to the immunogenic carrier has as effect the stimulation of the immune response against said saccharide, without inducing an immune response against the said immunogenic carrier.
  • a pure saccharide of general formula ( I ) contains a protective immunogenic glycan epitope and is able to induce a protective immune response against S. pneumoniae serotype 8 bacteria in a human and/or animal host.
  • the saccharide of general formula ( I ) elicits antibodies that are cross-reacting with the S. pneumoniae serotype 8 capsular polysaccharide, recognize specifically S. pneumoniae serotype 8 bacteria and opsonize them for killing by phagocytes.
  • the present invention provides a saccharide of general formula ( I ) V*-U x+3 -U x+2 -U x+1 -U x -O-L-NH 2 ( I ) wherein
  • the linker -L- is defined as a linker and is part of the fragment -O-L-NH 2 .
  • the linker -L- is bound to an oxygen atom and to the nitrogen atom of the NH 2 -group. It is preferred that at least two carbon atoms of the linker are between the oxygen atom and the NH 2 -group, like -O-C-C-NH 2 .
  • the linker -L- can be an aliphatic chain, wherein the aliphatic chain can optionally include an aromatic chain inserted in it, or a number of heteroatoms oscillating from 0 to 10.
  • the linker L preferably contains between 2 and 40 carbon atoms (including the carbon atoms of optional side chains), more preferably between 2 and 30, more preferably between 2 and 20, more preferably between 2 and 14, more preferably between 2 and 12, and still more preferably between 2 and 10 carbon atoms.
  • the shortest atom chain between the oxygen atom (i.e. the oxygen of -O-L-NH 2 ) and the NH 2 -group consists preferably of 2 to 14 atoms, more preferably of 2 to 12 atoms, more preferably of 2 to 10 atoms, more preferably of 2 to 8 atoms.
  • the shortest chain (which is the shortest possible connection between the oxygen at the anomeric center and the NH 2 -group) consists of 2 to 6 atoms, these are preferably carbon atoms.
  • the shortest chain consists of 4 to 8 atoms, the chain may contain 1, 2 or 3 heteroatoms selected from O, N and S.
  • the shortest chain consists of 9 to 14 atoms, the chain may contain 1, 2, 3, 4, 5, or 6 heteroatoms selected from O, N and S.
  • linker -L- or the shortest chain is fully or partially fluorinated.
  • the linker -L- may contain a 3-membered or a 4-membered or a 5-membered or a 6-membered saturated carbocycle or a 5-membered partly unsaturated (and not aromatic) carbocycle or a 4-membered or a 5-membered or a 6-membered saturated oxygen heterocycle or a 4-membered or a 5-membered or a 6-membered saturated nitrogen heterocycle or a 6-membered aromatic carbocycle.
  • the linker -L- may also contain amide (-NH-CO-, -CO-NH-) and/or urea (-NH-CO-NH-) residues and preferably only one amide or urea residue.
  • the linker may also contain substituents and preferably two substituents, such as R 10 and R 11 , or four substituents such as R 10 , R 11 , R 15 and R 14 , which have the meanings as defined herein and which are preferably selected from: -F, -Cl, -CH 3 , -C 2 H 5 , -C 3 H 7 , -C 5 H 9 , -C 6 H 13 , -OCH 3 , -OC 2 H 5 , -CH 2 F, -CHF 2 , -CF 3 , -C(O)-NH 2 , -SCH 3 , -SC 2 H 5 , -NHC(O)CH 3 , -N(CH 3 ) 2 , and -
  • linker -L- is fluorinated, more than two substituents -F are preferred.
  • the linker -L- is selected from: -CH 2 -, -(CH 2 ) 2 -, -(CH 2 ) 3 -, -(CH 2 ) 4 -, -(CH 2 ) 5 -, -(CH 2 ) 6 -, -(CH 2 ) 7 -, -(CH 2 ) 8 -, -(CH 2 ) 9 -, -(CH 2 ) 10 -, -CF 2 -, -(CF 2 ) 2 -, -(CF 2 ) 3 -, -(CF 2 ) 4 -, -(CF 2 ) 5 -, -(CF 2 ) 6 -, -(CF 2 ) 7 -, -(CF 2 ) 8 -, -(CF 2 ) 9 -, -(CF 2 ) 10 -, -(CH 2 ) 2 -O-(CH 2 ) 2 - -CH 2 -O-(CH 2
  • the saccharides of the present invention bear basic and/or acidic substituents and they may form salts with organic or inorganic acids or bases.
  • acids for such acid addition salt formation are hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, oxalic acid, malonic acid, salicylic acid, p-aminosalicylic acid, malic acid, fumaric acid, succinic acid, ascorbic acid, maleic acid, sulfonic acid, phosphonic acid, perchloric acid, nitric acid, formic acid, propionic acid, gluconic acid, lactic acid, tartaric acid, hydroxymaleic acid, pyruvic acid, phenylacetic acid, benzoic acid, p -aminobenzoic acid, p -hydroxybenzoic acid, methanesulfonic acid, ethanesulfonic acid, nitrous acid, hydroxyethanesulfonic acid, ethylenesulfonic acid, p -toluenesulfonic acid, naphthylsulfonic acid, sulfonic
  • Suitable inorganic or organic bases are, for example, NaOH, KOH, NH 4 OH, tetraalkylammonium hydroxide, lysine or arginine and the like. Salts may be prepared in a conventional manner using methods well known in the art, for example by treatment of a solution of the compound of the general formula ( I ) with a solution of a base, selected out of the group mentioned above.
  • the saccharides of general ( I ) are not containing -O-O- bonds and or sugar fragments (U x , U x+1 , U x+2 , U x+3 ) connected or bound to each other via their anomeric or C-1 carbons.
  • the stereochemistry of the glycosidic bond is the stereochemistry indicated for the anomeric centre of the sugar fragment in the general formula. Hence, the stereochemistry of the anomeric centre for sugar fragment U 1 and U 5 is ⁇ , for sugar fragment U 2 and U 6 is ⁇ , for sugar fragment U 3 and U 7 is ⁇ and for sugar fragment U 4 is ⁇ .
  • the saccharide of general formula ( I ) contains a protective immunogenic epitope and is able to induce a protective immune response against S. pneumoniae serotype 8 bacteria in a human and/or animal host.
  • the saccharide of general formula ( I ) elicits antibodies that are cross-reacting with the S. pneumoniae serotype 8 capsular polysaccharide, recognize specifically S. pneumoniae serotype 8 bacteria and opsonize them for killing by phagocytes.
  • the compounds of the invention according to general formula ( I ) have the advantage that these are pure synthesized compounds, which can be easily manufactured in accordance with GMP regulations, while isolated mixtures of saccharides as, for instance, disclosed in WO 9640225 A1 , are always not fully characterized mixtures with varying composition of the oligosaccharides depending on the source of isolation so that it is problematic to comply with the GMP regulations.
  • the vaccine of the present invention contains most preferably only one single compound of the general formula ( I ) or any other general formula ( I-a ) - ( I-c ), ( II ), ( II-a )-( II-g ), ( III ), ( III-a )-( III-g ), ( IV ), ( IV-a )-( IV-g ), ( V ), ( V-a ) - ( V-c ), ( VI ), ( VI-a )-( VI-c ), ( VII ), ( VII-a )-( VII-c ), ( VIII ), ( VIII-a )-( VIII-c ), ( IX ) or ( IX-a )-( IX-c ) disclosed herein and most preferably only one of the compounds 10 , 18 - 2 2 , 55 , 57 , 60 , and 62 - 89 bound to an immunogenic carrier, preferably a carrier protein and more preferably CRM 197 .
  • an immunogenic carrier preferably a carrier
  • the compounds of the general formulae ( I ) or any other general formula ( I-a )-( I-c ), ( II ), ( II-a )-( II-g ), ( III ), ( III-a )-( III-g ), ( IV ), ( IV-a )-( IV-g ), ( V ), ( V-a )-( V-c ), ( VI ), ( VI-a )-( VI-c ), ( VII ), ( VII-a )-( VII-c ), ( VIII ), ( VIII-a )-( VIII-c ), ( IX ) or ( IX-a )-( IX-c ) are used for the preparation of well defined, well characterized and pure vaccines containing only one synthetically synthesized and well characterized trisaccharide, tetrasaccharide, pentasaccharide, or hexasaccharide preferably linked to an immunogenic carrier, preferably a carrier protein and more preferably CRM 197
  • the vaccines of the present invention contain only one synthetically synthesized compound of general formulae (I) or any other general formula ( I-a )-( I-c ), ( II ), ( II-a )-( II-g ), ( III ), ( III-a )-( III-g ), ( IV ), ( IV-a )-( IV-g ), ( V ), ( V-a )-( V-c ), ( VI ), ( VI-a )-( VI-c ), ( VII ), ( VII-a )-( VII-c ), ( VIII ), ( VIII-a )-( VIII-c ), ( IX ) or ( IX-a )- (IX-c) preferably linked to an immunogenic carrier, preferably a carrier protein and more preferably CRM 197 .
  • an immunogenic carrier preferably a carrier protein and more preferably CRM 197 .
  • Such vaccines cause fewer side effects and/or non-protective immune responses in comparison to vaccines containing isolated (and not synthesized) mixtures of saccharides obtained by non-selective cleavage of the capsular polysaccharide of Streptococcus pneumoniae serotype 8.
  • inventive vaccines can be easier manufactured in accordance with the GMP regulations than the vaccines containing isolated mixtures of non-selectively cleaved capsular polysaccharides and are easier characterized, which makes stability and purity control easier as well as detection of kind and amount of impurities.
  • residue R # represents -COOH.
  • An embodiment of the present invention is directed to a saccharide of general formula (I) V*-U x+3 -U x+2 -U x+1 -U x -O-L-NH 2 (I) wherein V*- represents H-U x+1 -U x - and x, L, U x , U x+1 , U x+2 ,U x+3 and R # have the meanings as defined herein.
  • an embodiment of the present invention is directed to a hexasaccharide of general formula (II) H-U x+1 -U x -U x+3 -U x+2 -U x+1 -U x -O-L-NH 2 (II) wherein x is an integer selected from 1, 2, 3 and 4;
  • a hexasaccharide of general formula ( II-a ) wherein R # and L have the meanings defined herein; and a saccharide of general formula ( II-b ) wherein R # and L have the meanings defined herein; and a saccharide of general formula ( II-c ) wherein R # and L have the meanings defined herein; and a saccharide of general formula ( II-d ) wherein R # and L have the meanings defined herein is also preferred.
  • Another preferred embodiment is directed to a saccharide of general formula ( I ) V*-U x+3 -U x+2 -U x+1 -U x -O-L-NH 2 (I) wherein V*- represents H-U x - and x, L, U x , U x+1 , U x+2 , U x+3 and R # have the meanings as defined herein.
  • Another preferred embodiment is directed to a pentasaccharide of general formula (III) H-U x -U x+3 -U x+2 -U x+1 -U x -O-L-NH 2 ( III )
  • the residue R # represents -COOH.
  • a pentasaccharide of general formula ( III-a ) wherein R # and L have the meanings defined herein; and a saccharide of general formula ( III-b ) wherein R # and L have the meanings defined herein; and a saccharide of general formula ( III-c ) wherein R # and L have the meanings defined herein; and a saccharide of general formula ( III-d ) wherein R # and L have the meanings defined herein is also preferred.
  • a further preferred embodiment according to the present invention is directed to a saccharide of general formula ( I ) V*-U x+3 -U x+2 -U x+1 -U x -O-L-NH 2 ( I ) wherein V*- represents H- and x, L, U x , U x+1 , U x+2 , U x+3 and R # have the meanings as defined herein.
  • a further preferred embodiment according to the present invention is directed to a tetrasaccharide of general formula ( IV ) H-U x+3 -U x+2 -U x+1 -U x -O-L-NH 2 ( IV )
  • the residue R # represents -COOH.
  • a tetrasaccharide of general formula ( IV-a ) wherein R # and L have the meanings defined herein; and a saccharide of general formula ( IV-b ) wherein R # and L have the meanings defined herein; and a saccharide of general formula ( IV-c ) wherein R # and L have the meanings defined herein; and a saccharide of general formula ( IV-d ) wherein R # and L have the meanings defined herein is also preferred.
  • hexasaccharide ( II-a ), ( II-c ), ( II-d ), pentasaccharide ( III-a ), ( III-c ), ( III-d ) and tetrasaccharide ( IV-a ), ( IV-d ) is especially preferred.
  • Saccharides containing the protective immunogenic epitope ⁇ -D-Gl pc -(1 ⁇ 4)- ⁇ -D-Gl cp -(1 ⁇ 4)- ⁇ -D-Gal p are able to raise high titers of antibodies that recognize selectively S. pneumoniae type 8 and opsonize them for killing by phagocytes.
  • a saccharide of general formula ( I ) wherein x represents 2.
  • a saccharide of general formula ( VI ) V*-U 5 -U 4 -U 3 -U 2 -O-L-NH 2 ( VI ) wherein
  • the linker -L- is selected from: -L a -, -L a -L e -, -L a -L b -L e -, -L a -L d -L e -; wherein
  • the saccharide according to the present invention is selected from the group consisting of:
  • a pure saccharide of general formula ( VII ) contains the protective immunogenic glycan epitope ⁇ -D-Gl pc -(1 ⁇ 4)- ⁇ -D-Gl cp- (1 ⁇ 4)- ⁇ -D-Gal p and is able to induce a protective immune response against S. pneumoniae serotype 8 bacteria in a human and/or animal host.
  • the saccharide of general formula ( VII ) containing the protective immunogenic glycan epitope ⁇ -D-Gl pc -(1 ⁇ 4)- ⁇ -D-Gl cp -(1 ⁇ 4)- ⁇ -D-Gal p elicits antibodies that are cross-reacting with the S. pneumoniae serotype 8 capsular polysaccharide, recognize specifically S. pneumoniae serotype 8 bacteria and opsonize them for killing by phagocytes.
  • Another aspect of the present invention is directed to a saccharide of general formula ( VII ) S*-U 5 -U 4 -U 3 -S-O-L-NH 2 ( VII ) wherein
  • the linker -L- is defined as a linker and is part of the fragment -O-L-NH 2 .
  • the linker -L- is bound to an oxygen atom and to the nitrogen atom of the NH 2 -group. It is preferred that at least two carbon atoms of the linker are between the oxygen atom and the NH 2 -group, like -O-C-C-NH 2 .
  • the linker -L- can be an aliphatic chain, wherein the aliphatic chain can optionally include an aromatic chain inserted in it, or a number of heteroatoms oscillating from 0 to 10.
  • the linker L preferably contains between 2 and 40 carbon atoms (including the carbon atoms of optional side chains), more preferably between 2 and 30, more preferably between 2 and 20, more preferably between 2 and 14, more preferably between 2 and 12, and still more preferably between 2 and 10 carbon atoms.
  • the shortest atom chain between the oxygen atom (i.e. the oxygen of -O-L-NH 2 ) and the NH 2 -group consists preferably of 2 to 14 atoms, more preferably of 2 to 12 atoms, more preferably of 2 to 10 atoms, more preferably of 2 to 8 atoms.
  • the shortest chain (which is the shortest possible connection between the oxygen at the anomeric center and the NH 2 -group) consists of 2 to 6 atoms, these are preferably carbon atoms.
  • the shortest chain consists of 4 to 8 atoms, the chain may contain 1, 2 or 3 heteroatoms selected from O, N and S.
  • the shortest chain consists of 9 to 14 atoms, the chain may contain 1, 2, 3, 4, 5, or 6 heteroatoms selected from O, N and S.
  • linker -L- or the shortest chain is fully or partially fluorinated.
  • the linker -L- may contain a 3-membered or a 4-membered or a 5-membered or a 6-membered saturated carbocycle or a 5-membered partly unsaturated (and not aromatic) carbocycle or a 4-membered or a 5-membered or a 6-membered saturated oxygen heterocycle or a 4-membered or a 5-membered or a 6-membered saturated nitrogen heterocycle or a 6-membered aromatic carbocycle.
  • the linker -L- may also contain amide (-NH-CO-, -CO-NH-) and/or urea (-NH-CO-NH-) residues and preferably only one amide or urea residue.
  • the linker may also contain substituents and preferably two substituents such as R 10 and R 11 or four substituents such as R 10 , R 11 , R 15 and R 14 , which have the meanings as defined herein and which are preferably selected from: -F, -CI, -CH 3 , -C 2 H 5 , -C 3 H 7 , -C 5 H 9 , -C 6 H 13 , -OCH 3 , -OC 2 H 5 , -CH 2 F, -CHF 2 , -CF 3 , -C(O)-NH 2 , -SCH 3 , -SC 2 H 5 , -NHC(O)CH 3 , -N(CH 3 ) 2 , and -N(C 2
  • the linker -L- is selected from: -CH 2 -, -(CH 2 ) 2 -, -(CH 2 ) 3 -, -(CH 2 ) 4 -, -(CH 2 ) 5 -, -(CH 2 ) 6 -, -(CH 2 ) 7 -, -(CH 2 ) 8 -, -(CH 2 ) 9 -, -(CH 2 ) 10 -, -CF 2 -, -(CF 2 ) 2 -, -(CF 2 ) 3 -, -(CF 2 ) 4 -, -(CF 2 ) 5 -, -(CF 2 ) 6 -, -(CF 2 ) 7 -, -(CF 2 ) 8 --(CF 2 ) 9 -, -(CF 2 ) 10 -, -(CH 2 ) 2 -O-(CH 2 ) 2 -, -CH 2 -O-(CH 2 ) 2
  • the saccharides of the present invention bear basic and/or acidic substituents and they may form salts with organic or inorganic acids or bases.
  • acids for such acid addition salt formation are hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, oxalic acid, malonic acid, salicylic acid, p-aminosalicylic acid, malic acid, fumaric acid, succinic acid, ascorbic acid, maleic acid, sulfonic acid, phosphonic acid, perchloric acid, nitric acid, formic acid, propionic acid, gluconic acid, lactic acid, tartaric acid, hydroxymaleic acid, pyruvic acid, phenylacetic acid, benzoic acid, p-aminobenzoic acid, p-hydroxybenzoic acid, methanesulfonic acid, ethanesulfonic acid, nitrous acid, hydroxyethanesulfonic acid, ethylenesulfonic acid, p-toluenesulfonic acid, naphthylsulfonic acid, sulfanilic
  • Suitable inorganic or organic bases are, for example, NaOH, KOH, NH 4 OH, tetraalkylammonium hydroxide, lysine or arginine and the like. Salts may be prepared in a conventional manner using methods well known in the art, for example by treatment of a solution of the compound of the general formula ( I ) with a solution of a base, selected out of the group mentioned above.
  • the saccharides of general ( VII ) are not containing -O-O- bonds and or sugar fragments (U 2 , U 3 , U 4 , U 5 ) connected or bound to each other via their anomeric or C-1 carbons.
  • the stereochemistry of the glycosidic bond is the stereochemistry indicated for the anomeric centre of the sugar fragment in the general formula. Hence, the stereochemistry of the anomeric centre for sugar fragment U 1 and U 5 is ⁇ , for sugar fragment U 2 and U 6 is ⁇ , for sugar fragment U 3 and U 7 is ⁇ and for sugar fragment U 4 is ⁇ .
  • a saccharide of general formula ( VII ) wherein -S- represents -S a - and S*- represents S* a -.
  • a saccharide of general formula ( VIII ) S a *-U 5 -U 4 -U 3 -S a -O-L-NH 2 ( VIII ) wherein
  • a saccharide of general formula ( VII ) wherein -S- represents -S b - and S*- represents S* b -.
  • a saccharide of general formula ( IX ) S b *-U 5 -U 4 -U 3 -S b -O-L-NH 2 ( IX ) wherein
  • the linker -L- is selected from: -L a -, -L a -L e -, -L a -L b -L e -, -L a -L d -L e -; wherein
  • the saccharide according to the present invention is elected from the group consisting of:
  • a saccharide according to the present invention can be synthesized via several synthetic routes.
  • a saccharide according to the present invention can be assembled starting from thioglycoside building blocks BB2 (precursor for the sugar fragment U 1 , U 5 , U 2 and U 6 ), BB3 (precursor for the sugar fragment U 2 and U 6 ), BB4 (precursor for the sugar fragment U 3 and U 7 ) and BB5 (precursor for the sugar fragment U 4 ) and functionalized solid support BB1 (Angew. Chem. Int. Ed. 2013, 52, 5858.) (see Scheme 1 ) by automated solid phase synthesis.
  • solution-phase synthesis of oligosaccharides can be used for accessing saccharides of general formula ( I ).
  • disaccharidic building blocks such as BB6 and BB7 are preferably used as elongating units.
  • Alcohol BB9 can be further subjected to glycosylation reaction with disaccharide BB6 to provide tetrasaccharide BB8 that can be directly used as elongating unit.
  • the hydroxyl group at the C-4 position of the ⁇ -glucoside moiety can be protected with an orthogonal Fmoc protecting group that enables selective removal in anticipation of further glycosylation reactions.
  • the ⁇ -glucoside moiety constitutes the terminal monosaccharide at the non-reducing
  • protection of the hydroxyl group at the C-4 position as a benzoate ester will expedite the synthesis.
  • the synthetic oligosaccharides were printed on N -hydroxysuccinimide-functionalized glass slides and incubated with either a rabbit antiserum against the native Sp8 bacterium (type 8 antiserum, SSI Diagnostica, Figure 4 (D) ) or combined sera of humans vaccinated with Pneumovax23® ("007sp", National Institute for Biological Standards and Control, Figure 4 (B) , (C) ) . While the rabbit antiserum revealed recognition of all printed tetrasaccharides ( Figure 4 (D ) , the antiserum from human vaccinated with Pneumovax23® recognized only compounds 19, 20, 22 and 18 (see Figures 4 (B) and 4 (C) ) .
  • Microarray analysis enabled also the evaluation of the binding of the mAb 28H11 (a protective murine IgM) to saccharides 19, 20, 21 and 22 (see Figure 12 ) .
  • MAb 28H11 is a murine IgM that has been raised against native S. pneumoniae type 8 CPSs and protects mice from infection with live S. pneumoniae type 8 pneumococci in various settings ( Yano and Pirofski (2011), Clin. Vaccine Immunol., 18 (1), 59-66 ).
  • Glycan microarray analysis revealed a robust interaction of mAb 28H11 with saccharide 19 that was specific to S. pneumoniae type 8, as shown by the ablation of binding by native S. pneumoniae type 8 CPSs of up to 10 ⁇ g/mL (see Figure 12 ) .
  • Another aspect of the present invention is directed to a conjugate comprising a synthetic saccharide of general formula ( I ) covalently bound or covalently linked to an immunogenic carrier through the nitrogen atom of the -O-L-NH 2 group.
  • another aspect of the present invention is directed to a saccharide of any of the general formulae ( I ) , ( I-a ) - ( I-c ), ( II ), ( II-a )-( II-g ), ( III ), ( III-a )-( III-g ), ( IV ), ( IV-a ) - ( IV-g ), ( V ), ( V-a )-( V-c ), ( VI ), ( VI-a )-( VI-c ), ( VII ), ( VII-a )-( VII-c ), ( VIII ), ( VIII-a )-( VIII-c ), ( IX ), and ( IX-a )-( IX-c ) conjugated with an immunogenic carrier through the nitrogen
  • a conjugate comprising a synthetic saccharide of the general formula ( I ), ( I-a )-( I-c ), ( II ), ( II-a )-( II-g ), ( III ), ( III-a )-( III-g ), ( IV ), ( IV-a )-( IV-g ), ( V ), ( V-a )-( V-c ), ( VI ), ( VI-a )-( VI-c ), ( VII ), ( VII-a )-( VII-c ), ( VIII ), ( VIII-a )-( VIII-c ), ( IX ), or ( IX-a )-( IX-c ) covalently bound or covalently linked to an immunogenic carrier through the nitrogen atom of the -O-L-NH 2 group is also defined as a conjugate obtained by reacting a saccharide of any of the general formulae ( I ), ( I-a )-( I-c ), ( II ), ( II-a )
  • TI-2 antigens are antigens, which are recognized only by mature B cells through the cross linking of surface exposed immunoglobulin receptors. Without T cell help, no immunological memory is generated and neither isotype switching from IgM to other IgG subclasses, nor B cells affinity maturation occurs.
  • saccharides are known poor immunogens in humans due to the structural homology to human glycolipids and glycoproteins. Due to their poor immunogenic properties, saccharides manifest poor ability to produce both antibody production by B cells, as well as the formation of memory cells, features which are essential for the production of potent vaccines.
  • Said conjugate consists of at least one synthetic saccharide of the general formula ( I ), ( I-a )-( I-c ), ( II ), ( II-a )-( II-g ), ( III ), ( III-a )-( III-g ), ( IV ), ( IV-a )-( IV-g ), ( V ), ( V-a )-( V-c ), ( VI ), ( VI-a )-( VI-c ), ( VII ), ( VII-a )-( VII-c ), ( VIII ), ( VIII-a )-( VIII-c ), ( IX ), or ( IX-a )-( IX-c ) and an immunogenic carrier to which the at least one saccharide of the general formula ( I ), ( I-a )-( I-c ), ( II ), ( II-a )-( II-g ), ( III ), ( III-a )-( III-g ), ( IV ), ( IV-a )
  • the term "immunogenic carrier” is defined as a structure, which is conjugated to the saccharide to form a conjugate that presents an increased immunogenicity in comparison with the saccharide per se.
  • the conjugation of a saccharides of the general formula ( I ), ( I-a )-( I-c ), ( II ), ( II-a )-( II-g ), ( III ), ( III-a )-( III-g ), ( IV ), ( IV-a )-( IV-g ), ( V ), ( V-a )-( V-c ), ( VI ), ( VI-a )-( VI-c ), ( VII ), ( VII-a )-( VII-c ), ( VIII ), ( VIII-a )-( VIII-c ), ( IX ), or ( IX-a )-( IX-c ) to the immunogenic carrier has as effect the stimulation of the immune response against the saccharide of the general formula ( I ), ( I-a )-
  • a carrier protein is a protein selected from the group comprising or consisting of: a diphtheria toxoid, a mutated diphtheria toxoid, a modified diphtheria toxoid, a mutated and modified diphtheria toxoid, a tetanus toxoid, a modified tetanus toxoid, a mutated tetanus toxoid, outer membrane protein (OMP), bovine serum albumin (BSA), keyhole limpet hemocyanine (KLH) or cholera toxoid (CT).
  • OMP outer membrane protein
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanine
  • CT cholera toxoid
  • toxoid refers to a bacterial toxin (usually an exotoxin), whose toxicity has been inactivated or suppressed either by chemical (formalin) or heat treatment, while other properties, typically immunogenicity, are maintained.
  • a mutated toxoid as used herein is a recombinant bacterial toxin, which has been amended to be less toxic or even non-toxic by amending the wild-type amino acid sequence. Such a mutation could be a substitution of one or more amino acids.
  • Such a mutated toxoid presents on its surface a functionality that can react with the functional group Y of the interconnecting molecule to provide a modified toxoid.
  • Said functionality is known to the person skilled in the art and includes, but is not restricted to the primary amino functionality of a lysine residue that can react with activated esters, an isocyanate group or an aldehyde in presence of a reducing agent, to the carboxylate functionality of a glutamate or aspartate residue that can be activated by carbodiimides or to the thiol functionality of a cysteine residue.
  • Activated esters include, but are not restricted to N-( ⁇ -maleimidobutyryloxy) sulfosuccinimide ester (sulfo-GMBS), succinimidyl (4-iodoacetyl) aminobenzoate (sulfo-SIAB), succinimidyl-3-(bromoacetamido)propionate (SBAP), disuccinimidyl glutarate (DSG), disuccinimidyl adipate (DSA), 2-pyridyldithiol-tetraoxatetradecane- N -hydroxysuccinimide (PEG-4-SPDP), bis-(4-nitrophenyl) adipate and bis-(4-nitrophenyl) succinate (see Figure 1 ).
  • sulfo-GMBS N-( ⁇ -maleimidobutyryloxy) sulfosuccinimide ester
  • Preferred activated esters are disuccinimidyl adipate (DSA), disuccinimidyl glutarate (DSG), bis-(4-nitrophenyl) adipate and bis-(4-nitrophenyl) succinate.
  • the cysteine residue on the carrier protein can be converted to the corresponding dehydroalanine that can be further reacted with a suitable interconnecting molecule to provide modified carrier protein having on their surface the functional group X of the interconnecting molecule.
  • inventive saccharides described herein are conjugated to the non-toxic mutated diphtheria toxin CRM 197 presenting as a functionality a primary amine functionality of a lysine residue.
  • CRM 197 like wild-type diphtheria toxin is a single polypeptide chain of 535 amino acids (58 kD) consisting of two subunits linked by disulfide bridges having a single amino acid substitution of glutamic acid for glycine. It is utilized as a carrier protein in a number of approved conjugate vaccines for diseases such as Prevnar.
  • the carrier protein presents on its surface primary amino functionalities of lysine residues that are able to react with the functional group Y of the interconnecting molecule to provide modified carrier protein having on their surface said functional group X of the interconnecting molecule, which is able to react with the terminal amino group of the linker functionalizing the inventive saccharides.
  • Said functional group X of the interconnecting molecules is selected from the group comprising or consisting of maleimide; ⁇ -iodoacetyl; ⁇ -bromoacetyl; and N-hydroxysuccinimide ester (NHS), aldehyde, imidoester, carboxylic acid, alkyl sulfonate, sulfonyl chloride, epoxide, anhydride, carbonate (see Figure 2 ).
  • Preferred is a conjugate of general formula ( X ) [V*-U x+3 -U x+2 -U x+1 -U x -O-L-NH-W] m -CRM 197 ( X )
  • the linker -L- is selected from: -L a -, -L a -L e -, -L a -L b -L e -, and -L a -L d -L e -;
  • -L a - is selected from: -(CH 2 ) o -, -(CH 2 -CH 2 -O) o -C 2 H 4 -, -(CH 2 -CH 2 -O) o -CH 2 -;
  • -L b - represents -O-;
  • -L d - is selected from: -(CH 2 ) q -, -(CF 2 ) q -, -(CH 2 -CH 2 -O) q -C 2 H 4 -, and -(CH 2 -CH 2 -O) q -CH 2 -;
  • -L e - is selected from: -
  • -W- represents and a is an integer selected from 2, 3, 4, 5 and 6 is especially preferred.
  • -L a - is selected from: -(CH 2 ) o -, -(CH 2 -CH 2 -O) o -C 2 H 4 -, -(CH 2 -CH 2 -O) o -CH 2 -;
  • -L b - represents -O-;
  • -L d - is selected from: -(CH 2 ) q -, -(CF 2 ) q -, -(CH 2 -CH 2 -O) q -C 2 H 4 -, and -(CH 2 -CH 2 -O) q -CH 2 -;
  • the linker -L- is selected from: -L a -, -L a -L e -, -L a -L b -L e -, and -L a -L d -L e -;
  • -L a - is selected from: -(CH 2 ) o -, -(CH 2 -CH 2 -O) o -C 2 H 4 -, -(CH 2 -CH 2 -O) o -CH 2 -;
  • -L b - represents -O-;
  • -L d - is selected from: -(CH 2 ) q -, -(CF 2 ) q -, -(CH 2 -CH 2 -O) q -C 2 H 4 -, and -(CH 2 -CH 2 -O) q -CH 2 -;
  • -L e - is selected from: -
  • m is comprised between 2 and 18, more preferably between 5 and 15, even more preferably between 8 and 12.
  • Another aspect of the present invention is directed to a conjugate of general formula ( XII ) [S*-U 5 -U 4 -U 3 -S-O-L-NH-W] m -CRM 197 ( XII ) wherein
  • a further preferred conjugate is a conjugate of general formula ( XIII ) [S b *-U 5 -U 4 -U 3 -S b -O-L-NH-W] m -CRM 197 ( XIII ) wherein
  • the linker -L- is selected from: -L a -, -L a -L e -, -L a -L b -L e - and -L a -L d -L e -;
  • -L a - is selected from: -(CH 2 ) o -, -(CH 2 -CH 2 -O) o -C 2 H 4 -, -(CH 2 -CH 2 -O) o -CH 2 -;
  • -L b - represents -O-;
  • -L d - is selected from: -(CH 2 ) q -, -(CF 2 ) q -, -(CH 2 -CH 2 -O) q -C 2 H 4 -, and -(CH 2 -CH 2 -O) q -CH 2 -;
  • -L e - is selected from: -(
  • -W- represents and a is an integer selected from 2, 3, 4, 5 and 6 is especially preferred.
  • -L a - is selected from: -(CH 2 ) o -, -(CH 2 -CH 2 -O) o -C 2 H 4 -, -(CH 2 -CH 2 -O) o -CH 2 -;
  • -L b - represents -O-;
  • -L d - is selected from: -(CH 2 ) q -, -(CF 2 ) q -, -(CH 2 -CH 2 -O) q -C 2 H 4 -, and -(CH 2 -CH 2 -O) q
  • said immunogenic carrier is preferably a glycosphingolipid with immunomodulatory properties, and more preferably (2 S ,3 S ,4 R )-1-( ⁇ -D-galactopyranosyl)-2-hexacosanoylaminooctadecane-3,4-diol.
  • glycosphingolipid with immunomodulatory properties refers to a suitable glycosphingolipid capable of stimulating the immune system's response to a target antigen, but which does not in itself confer immunity as defined above.
  • Glycosphingolipids as used herein are compounds containing a carbohydrate moiety ⁇ -linked to a sphingolipid.
  • the carbohydrate moiety is a hexopyranose and most preferably is ⁇ -D-galactopyranose.
  • sphingolipids are a class of lipids containing a C18 amino alcohol connected via an amide bond to a fatty acid.
  • the C18 amino alcohol is preferably mono-, di- or polysubstituted with hydroxyl groups.
  • the C18 amino alcohol is phytosphingosine.
  • the fatty acid is preferably a monocarboxylic acid having a saturated alkyl chain of a number of carbons ranging from 16 to 28 and more preferably from 18 to 26.
  • Glycosphingolipids with immunomodulatory properties include, but they are not restricted to (2 S ,3 S ,4 R )-1-( ⁇ -D-galactopyranosyl)-2-hexacosanoylaminooctadecane-3,4-diol, which can stimulate natural killer (NK) activity and cytokine production by natural killer T (NKT) cells and exhibits potent antitumor activity in vivo ( Proc. Natl Acad. Sci. USA, 1998, 95, 5690 ).
  • the conjugates of the inventive saccharides with a glycosphingolipid with immunomodulatory properties have the advantage of being heat stable.
  • a functionality is introduced on the glycosphingolipid with immunomodulatory properties. Said functionality is prone to react directly with the terminal amino group of the linker of the inventive to provide conjugates of the saccharides or with the functional group Y of the interconnecting molecule to provide the modified glycosphingolipid with immunomodulatory properties.
  • the glycosphingolipid with immunomodulatory properties is functionalized with a functionality, which is prone of reacting with the terminal amino group of the saccharides or with the functional group Y of the interconnecting molecule.
  • a functionality prone to react with an amino group includes, but it is not restricted to activated ester, isocyanate group, aldehyde, epoxide, imidoester, carboxylic acid, alkyl sulfonate and sulfonyl chloride.
  • a functionality prone to react with the functional group Y of the interconnecting molecule so that to provide the modified glycosphingolipid with immunomodulatory properties presenting the functional group X of the interconnecting molecule includes, but it is not restricted to amine, alcohol, thiol, activated ester, isocyanate group, aldehyde, epoxide, vinyl, imidoester, carboxylic acid, alkyl sulfonate, sulfonyl chloride, vinyl group, alkynyl group and azido group.
  • the functionality introduced at the C-6 position of the carbohydrate moiety of the glycosphingolipid with immunomodulatory properties is selected from the group comprising or containing an amine, a thiol, an alcohol, a carboxylic acid, a vinyl, maleimide, ⁇ -iodoacetyl, ⁇ -bromoacetyl, N -hydroxysuccinimide ester (NHS), 2-pyridyldithiols.
  • Said functional group X of the interconnecting molecules is selected of the group comprising or consisting of maleimide, ⁇ -iodoacetyl, ⁇ -bromoacetyl, N- hydroxysuccinimide ester (NHS), aldehyde, carboxylic acid, epoxyde, alkyl sulfonate, sulfonyl chloride, anhydride, carbonate.
  • di( N -succinimidyl) adipate or bis(4-nitrophenyl) adipate is first reacted with a synthetic saccharide having a primary amino group.
  • Activated saccharide is subsequently condensed with a glycosphingolipid, which is modified at C-6 position by an interconnecting molecule having a terminal amino functionality in order to afford the conjugate ( Figure 3 (B) ) .
  • interconnecting molecule refers to a bifunctional molecule containing functional group X and functional group Y, wherein functional group X is capable of reacting with the terminal amino group on the linker -L- and the functional group Y is capable of reacting with a functionality present on the immunogenic carrier or on the solid support.
  • the bacterium containing in the capsular polysaccharide one of the above mentioned saccharide fragments is Streptococcus pneumoniae serotype 8.
  • Diseases associated with Streptococcus pneumoniae serotype 8 include pneumonia, meningitis, otitis media, bacteremia and acute exacerbation of chronic bronchitis, sinusitis, arthritis and conjunctivitis.
  • One aspect of the present invention relates to a pharmaceutical composition, especially a vaccine containing at least one synthetic saccharide according to the present invention and/or a pharmaceutical acceptable salt thereof and/or a conjugate comprising a saccharide according to the present invention covalently linked to an immunogenic carrier through the nitrogen atom of the -O-L-NH 2 group together with at least one pharmaceutical acceptable adjuvant, cryoprotectant, lyoprotectant, excipient and/or diluent.
  • said pharmaceutical composition or vaccine further comprises at least one of capsular polysaccharides and/or capsular polysaccharide fragments and/or protein conjugates thereof of Streptococcus pneumoniae bacteria selected from the group comprising or consisting of Streptococcus pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, preferably serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19F and 23F, and more preferably serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F and 33F.
  • Streptococcus pneumoniae bacteria selected from the group comprising or consisting of Streptococcus pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, preferably serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V
  • the vaccine may be prepared in the form of a suspension or may be lyophilized.
  • the suspension form may be stored frozen. In the lyophilized form, it is preferable to add one or more stabilizers.
  • Vaccination can be performed at any age.
  • the vaccine many be administered subcutaneously, by spray, by injection, orally, intraocularly, intratracheally or nasally.
  • the amount of vaccine of the invention to be administered a human or animal and the regime of administration can be determined in accordance with standard techniques well known to those of ordinary skill in the pharmaceutical and veterinary arts taking into consideration such factors as the particular antigen, the adjuvant (if present), the age, sex, weight, species and condition of the particular animal or human host, and the route of administration.
  • Another aspect of the present invention is directed to a method of inducing immune response against Streptococcus pneumoniae in a human and/or animal host, said method comprising administering of the saccharide according to the present invention and/or salt thereof and/or a a conjugate comprising a saccharide according to the present invention covalently linked to an immunogenic carrier through the nitrogen atom of the -O-L-NH 2 group and/or a mixture thereof or pharmaceutical composition thereof to said human and/or animal host.
  • a method of treating or preventing diseases caused by Streptococcus pneumoniae in a human and/or animal host according to the present invention comprises administering of at least one saccharide according to the present invention and/or salt thereof and/or a conjugate comprising a saccharide according to the present invention covalently linked to an immunogenic carrier through the nitrogen atom of the -O-L-NH 2 group and/or a mixture thereof or pharmaceutical composition thereof to said human and/or animal host.
  • compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like.
  • a suitable carrier diluent, or excipient
  • lyophilized saccharides of the invention can ultimately be reconstituted with a liquid component to give material suitable for administration to a said human and/or animal host. The reconstitution will typically take place at the point of use.
  • a saccharide of the invention and an oil-in-water emulsion adjuvant or a buffer solution of an adjuvant may be kept separately in a packaged or distributed vaccine kit, ready for final formulation at the time of use.
  • the lyophilized component of the invention may include a stabilizer such as lactose, sucrose and/or mannitol, as well as mixtures thereof. Using a sucrose/mannitol mixture can speed up the drying process.
  • a lyophilized component may also include sodium chloride. Soluble components in the lyophilized material will be retained in the composition after reconstitution, and so final liquid vaccines may thus contain lactose and/or sucrose.
  • Formulation of the vaccines of the present invention can be accomplished using methods known by the art. Obviously, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form.
  • the vaccine compositions of the present invention may contain one or more adjuvants.
  • adjuvant refers to an immunological adjuvant i.e. a material used in a vaccine composition that modifies or augments the effects of said vaccine by enhancing the immune response to a given antigen contained in the vaccine without being antigenically related to it.
  • immunological adjuvants include, but are not restricted to oil emulsions (e.g. Freund's adjuvant), saponins, aluminium or calcium salts (e.g. alum), non-ionic block polymer surfactants, ⁇ -galactosylceramide and many others.
  • compositions of the invention can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired.
  • Compositions of the invention may include an antimicrobial, particularly when packaged in multiple dose formats. Antimicrobials such as thiomersal and 2-phenoxyethanol are commonly found in vaccines, but it is preferred to use either a mercury-free preservative or no preservative at all.
  • inventive vaccines may include a temperature protective agent.
  • a temperature protective agent examples include glycerin, propylene glycol, and/or polyethylene glycol (PEG).
  • compositions of the present invention are conveniently provided as liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions or viscous compositions that may be buffered to a selected pH.
  • Pharmaceutically acceptable carriers for liquid formulations may be aqueous or non-aqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic solutions, emulsions or suspensions, including saline and buffered media.
  • compositions of the invention may be maintained by the use of a buffer e.g. a Tris buffer, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate and triethanolamine buffer.
  • a buffer e.g. a Tris buffer, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate and triethanolamine buffer.
  • the isotonic agent may be an ionic isotonic agent such as a salt or a non-ionic isotonic agent such as a carbohydrate.
  • ionic isotonic agents include, but are not limited to NaCl, CaCl 2 , KCl and MgCl 2 .
  • non-ionic isotonic agents include but are not limited to sorbitol and glycerol.
  • the vaccine composition is formulated as a sterile liquid, pyrogene-free, phosphate-buffered physiological saline, with or without a preservative.
  • a pharmaceutically acceptable preservative can be employed to increase the shelf life of the compositions.
  • Benzyl alcohol may be suitable, although a variety of preservatives including, for example, parabens, thimerosal, chlorobutanol, or benzalkonium chloride may also be employed.
  • a suitable concentration of the preservative will be from 0.02% to 2% based on the total weight although there may be appreciable variation depending upon the agent selected.
  • compositions of the invention can be formulated as single dose vials, multidose vials or as pre-filled syringes.
  • the pharmaceutical composition is formulated in the form of a lyophilisate or liquid buffer solution.
  • the vaccine or pharmaceutical composition of the present invention is prepared in a conventional solid or liquid carrier or diluents and a conventional pharmaceutically-made adjuvant at suitable dosage level in a known way.
  • the preferred preparations and formulations are in administrable, form which is suitable for oral application. These administrable forms, for example, include pills, tablets, film tablets, coated tablets, capsules, powders and deposits. Forms other than oral administrable forms are also possible.
  • inventive vaccine or pharmaceutical composition may be administered by any appropriate means, including but not limited to inhalation, injection (intravenous, intraperitoneal, intramuscular, subcutaneous) by absorption through epithelial or mucocutaneous linings (oral mucosa, rectal and vaginal epithelial linings, nasopharyngial mucosa, intestinal mucosa); orally, rectally, transdermally, topically, intradermally, intragastrically, intracutaneously, intravaginally, intravasally, intranasally, intrabuccally, percutaneously, sublingually, or any other means available within the pharmaceutical arts.
  • the vaccine or pharmaceutical composition of the present invention containing at least one synthetic saccharide of any of general formulae ( I ), ( I-a )-( I-c ), ( II ), ( II-a )-( II-g ), ( III ), ( III-a )-( III-g ), ( IV ), ( IV-a )-( IV-g ), ( V ), ( V-a )-( V-c ), ( VI ), ( VI-a )-( VI-c ), ( VII ), ( VII-a ) - ( VII-c ), ( VIII ), ( VIII-a )-( VIII-c ), ( IX ), and ( IX-a )-( IX-c ), preferably the saccharides 10, 18, 19, 20, 21, 22, 55, 57, 60 and 62 or pharmaceutically acceptable salt thereof, or a a conjugate comprising a saccharide of general formula ( I ), ( I-a )-( I-c ), ( II ), ( II-a )
  • the active ingredient may be combined with any oral nontoxic pharmaceutically acceptable inert carrier, such as lactose, starch, sucrose, cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, talc, mannitol, ethyl alcohol (liquid forms) and the like.
  • suitable binders, lubricants, disintegrating agents and coloring agents may also be incorporated in the mixture.
  • Powders and tablets may be comprised of from about 5 to about 95 percent of the saccharide according to the present invention.
  • Suitable binders include starch, gelatin, natural sugars, corn sweeteners, natural and synthetic gums such as acacia, sodium alginate, carboxymethyl-cellulose, polyethylene glycol and waxes.
  • lubricants that may be mentioned for use in these dosage forms, boric acid, sodium benzoate, sodium acetate, sodium chloride, and the like.
  • Disintegrants include starch, methylcellulose, guar gum and the like. Sweetening and flavouring agents and preservatives may also be included where appropriate.
  • the vaccine or pharmaceutical composition of the present invention may be formulated in sustained release form to provide the rate controlled release of any one or more of the components or active ingredients to optimize the therapeutic effects.
  • Suitable dosage forms for sustained release include layered tablets containing layers of varying disintegration rates or controlled release polymeric matrices impregnated with the active components and shaped in tablet form or capsules containing such impregnated or encapsulated porous polymeric matrices.
  • Liquid form preparations include solutions, suspensions and emulsions. As an example may be mentioned water or water-propylene glycol solutions for parenteral injections or addition of sweeteners and opacifiers for oral solutions, suspensions and emulsions. Liquid form preparations may also include solutions for intranasal administration.
  • Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be in combination with a pharmaceutically acceptable carrier such as inert compressed gas, e.g. nitrogen.
  • a pharmaceutically acceptable carrier such as inert compressed gas, e.g. nitrogen.
  • a low melting wax such as a mixture of fatty acid glycerides such as cocoa butter is first melted, and the active ingredient is dispersed homogeneously therein by stirring or similar mixing. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool and thereby solidifies.
  • solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration.
  • liquid forms include solutions, suspensions and emulsions.
  • the inventive vaccine or pharmaceutical composition containing at least one synthetic saccharide of any of general formulae ( I ), ( I-a )-( I-c ), ( II ), ( II-a )-( II-g ), ( III ), ( III-a )-( III-g ), ( IV ), ( IV-a )-( IV-g ), ( V ), ( V-a )-( V-c ), ( VI ), ( VI-a )-( VI-c ), ( VII ), ( VII-a )-( VII-c ), ( VIII ), ( VIII-a )-( VIII-c ), ( IX ), and ( IX-a )-( IX-c ), preferably the saccharides 10, 18, 19, 20, 21, 22, 55, 57, 60 and 62 - 89 or pharmaceutically acceptable salt thereof, or a conjugate comprising a saccharide covalently linked or covalently bound to an immunogenic carrier through the nitrogen atom of the -O-L-NH 2 group may also
  • capsule refers to a special container or enclosure made of methyl cellulose, polyvinyl alcohols, or denatured gelatins or starch for holding or containing compositions comprising the active ingredients.
  • Hard shell capsules are typically made of blends of relatively high gel strength bone and pork skin gelatins.
  • the capsule itself may contain small amounts of dyes, opaquing agents, plasticizers and preservatives.
  • Tablet means compressed or moulded solid dosage form containing the active ingredients with suitable diluents.
  • the tablet can be prepared by compression of mixtures or granulations obtained by wet granulation, dry granulation or by compaction well known to a person skilled in the art.
  • Oral gels refer to the active ingredients dispersed or solubilized in a hydrophilic semi-solid matrix.
  • Powders for constitution refer to powder blends containing the active ingredients and suitable diluents which can be suspended in water or juices.
  • Suitable diluents are substances that usually make up the major portion of the composition or dosage form. Suitable diluents include sugars such as lactose, sucrose, mannitol and sorbitol, starches derived from wheat, corn rice and potato, and celluloses such as microcrystalline cellulose.
  • the amount of diluents in the composition can range from about 5 to about 95% by weight of the total composition, preferably from about 25 to about 75%, more preferably from about 30 to about 60% by weight, and most preferably from about 40 to 50% by weight.
  • disintegrants refers to materials added to the composition to help it break apart (disintegrate) and release the medicaments.
  • Suitable disintegrants include starches, "cold water soluble" modified starches such as sodium carboxymethyl starch, natural and synthetic gums such as locust bean, karaya, guar, tragacanth and agar, cellulose derivatives, such as methylcellulose and sodium carboxymethylcellulose, microcrystalline celluloses and cross-linked microcrystalline celluloses such as sodium croscarmellose, alginates such as alginic acid and sodium alginate, clays such as bentonites, and effervescent mixtures.
  • the amount of disintegrant in the composition can range from about 1 to about 40% by weight of the composition, preferably 2 to about 30% by weight of the composition, more preferably from about 3 to 20% by weight of the composition, and most preferably from about 5 to about 10% by weight.
  • Binders characterize substances that bind or "glue” powders together and make them cohesive by forming granules, thus serving as the "adhesive" in the formulation. Binders add cohesive strength already available in the diluents or bulking agent. Suitable binders include sugars such as sucrose, starches derived from wheat, corn rice and potato; natural gums such as acacia, gelatin and tragacanth; derivatives of seaweed such as alginic acid, sodium alginate and ammonium calcium alginate; cellulosic materials such as methylcellulose and sodium carboxymethylcellulose and hydroxypropyl-methylcellulose; polyvinylpyrrolidone; and inorganics such as magnesium aluminum silicate.
  • the amount of binder in the composition can range from about 1 to 30% by weight of the composition, preferably from about 2 to about 20% by weight of the composition, more preferably from about 3 to about 10% by weight, even more preferably from about 3 to about 6% by weight.
  • Lubricant refers to a substance added to the dosage form to enable the tablet, granules, etc. after it has been compressed, to release from the mold or die by reducing friction or wear.
  • Suitable lubricants include metallic stearates such as magnesium stearate, calcium stearate or potassium stearate; stearic acid; high melting point waxes; and water soluble lubricants such as sodium chloride, sodium benzoate, sodium acetate, sodium oleate, polyethylene glycols and d'I-leucine. Lubricants are usually added at the very last step before compression, since they must be present on the surfaces of the granules and in between them and the parts of the tablet press.
  • the amount of lubricant in the composition can range from about 0.05 to about 15% by weight of the composition, preferably 0.2 to about 5% by weight of the composition, more preferably from about 0.3 to about 3%, and most preferably from about 0.3 to about 1.5% by weight of the composition.
  • Glidents are materials that prevent caking and improve the flow characteristics of granulations, so that flow is smooth and uniform.
  • Suitable glidents include silicon dioxide and talc.
  • the amount of glident in the composition can range from about 0.01 to 10% by weight of the composition, preferably 0.1% to about 7% by weight of the total composition, more preferably from about 0.2 to 5% by weight, and most preferably from about 0.5 to about 2% by weight.
  • Colouring agents are excipients that provide coloration to the composition or the dosage form. Such excipients can include food grade dyes and food grade dyes adsorbed onto a suitable adsorbent such as clay or aluminium oxide.
  • the amount of the colouring agent can vary from about 0.01 to 10% by weight of the composition, preferably from about 0.05 to 6% by weight, more preferably from about 0.1 to about 4% by weight of the composition, and most preferably from about 0.1 to about 1%.
  • a suitable vaccine composition comprising at least one saccharide of any one of the general formulae ( I ), ( I-a )-( I-c ), ( II ), ( II-a )-( II-g ), ( III ), ( III-a )-( III-g ), ( IV ), ( IV-a )-( IV-g ), ( V ), ( V-a )-( V-c ), ( VI ), ( VI-a )-( VI-c ), ( VII ), ( VII-a )-( VII-c ), ( VIII ), ( VIII-a )-( VIII-c ), ( IX ), and ( IX-a )-( IX-c ), preferably the saccharides 10, 18, 19, 20, 21, 22, 55, 57, 60 and 62 - 89 or pharmaceutically acceptable salt thereof, or a conjugate comprising a saccharides 10, 18, 19, 20, 21, 22, 55, 57, 60 and 62 - 89 or pharmaceutically acceptable salt thereof, or a conjugate comprising
  • a therapeutically effective dosage of one conjugate according to the present invention or of one saccharide according to the present invention refers to that amount of the compound that results in an at least a partial immunization against a disease.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical, pharmacological, and toxicological procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutic effect is the therapeutic index.
  • the actual amount of the composition administered will be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgement of the prescribing physician.
  • the present invention refers also to an antibody against at least one synthetic saccharide according to the present invention.
  • the antibody is produced by the monoclonal hybridoma.
  • the antibody is useful for diagnostics, prophylaxis, and treatment of pneumonia, meningitis, otitis media, bacteremia and acute exacerbation of chronic bronchitis, sinusitis, arthritis and conjunctivitis.
  • Another embodiment of the present invention concerns the use of the antibody for manufacture of medicaments or devices for diagnosis, prophylaxis, and treatment of pneumonia, meningitis, otitis media, bacteremia and acute exacerbation of chronic bronchitis, sinusitis, arthritis and conjunctivitis caused by S. pneumoniae, and preferably by S. pneumoniae serotype 8.
  • antibody encompasses polyclonal and monoclonal antibody preparations, as well as preparations including hybrid antibodies, F(ab') 2 fragments, F(ab) molecules, single domain antibodies and functional fragments thereof, which exhibit immunological binding properties of the parent antibody molecule.
  • the antibody according to the invention may be polyclonal or monoclonal.
  • the saccharide of any one of general formulae ( I ), ( I-a )-( I-c ), ( II ), ( II-a )-( II-g ), ( III ), ( III-a )-( III-g ), ( IV ), ( IV-a )-( IV-g ), ( V ), ( V-a )-( V-c ), ( VI ), ( VI-a )-( VI-c ), ( VII ), ( VII-a )-( VII-c ), ( VIII ), ( VIII-a )-( VIII-c ), ( IX ), and ( IX-a )-( IX-c ), preferably the saccharides 10, 18, 19, 20, 21, 22, 55, 57, 60 or 62 - 89 or a conjugate comprising one of the above-mentioned saccharides covalently linked to an immunogenic carrier through the nitrogen atom of the -O-L-NH 2 group or the antibody thereof can be used for preparing a pharmaceutical composition, especially
  • the saccharide of the present invention or the antibody raised against and recognizing the saccharide according to the present invention can be used for the treatment or prevention of a disease caused by S. pneumoniae, preferably S. pneumoniae serotype 8.
  • the present invention refers to at least one saccharide of general formula ( I ), ( I-a )-( I-c ), ( II ), ( II-a )-( II-g ), ( III ), ( III-a )-( III-g ), ( IV ), ( IV-a )-( IV-g ), ( V ), ( V-a )-( V-c ), ( VI ), ( VI-a )-( VI-c ), ( VII ), ( VII-a )-( VII-c ), ( VIII ), ( VIII-a )-( VIII-c ), ( IX ), and ( IX-a )-( IX-c ) according to the invention or at least one antibody against at least one saccharide of the present invention for use in immunological assays for diagnosis of pneumonia, meningitis, otitis media, bacteremia and acute exacerbation of chronic bronchitis, sinusitis, arthritis and conjunctivitis caused by S. pneumoniae,
  • Such assays comprise, for instance, microarray and ELISA useful for diagnosis of diseases caused by S. pneumoniae, preferably S. pneumoniae serotype 8. Therefore, another aspect of the present invention refers to the use of any one of saccharides of formulae ( I ), ( I-a ) - ( I-c ), ( II ), ( II- )-( II-g ), ( III ), ( III-a )-( III-g ), ( IV ), ( IV-a )-( IV-g ), ( V ), ( V-a )-( V-c ), ( VI ), ( VI-a )-( VI-c ), ( VII ), ( VII-a )-( VII-c ), ( VIII ), ( VIII-a )-( VIII-c ), ( IX ), and ( IX-a )-( IX-c ) for diagnosis of diseases caused by S. pneumoniae, preferably S. pneumoniae serotype 8.
  • a method of identifying S. pneumoniae serotype 8 comprises the use of at least one saccharide of the present invention.
  • the synthetic saccharide of general formulae ( I ), ( I-a )-( I-c ), ( II ), ( II-a )-( II-g ), ( III ), ( III-a )-( III-g ), ( IV ), ( IV-a )-( IV-g ), ( V ), ( V-a ) - ( V-c ), ( VI ), ( VI-a )-( VI-c ), ( VII ), ( VII-a )-( VII-c ), ( VIII ), ( VIII-a )-( VIII-c ), ( IX ), and ( IX-a )-( IX-c ) or a mixture of such saccharides can be used as an analytical standard for immunoassays.
  • another aspect of the present invention is related to a solid support comprising at least one saccharide according to the present invention
  • This solid support is preferable a part of a diagnostic device.
  • the solid support and the diagnostic device are used for diagnosis of pneumonia, meningitis, otitis media, bacteremia and acute exacerbation of chronic bronchitis, sinusitis, arthritis and conjunctivitis caused by S. pneumoniae, preferably S. pneumoniae serotype 8, wherein the at least one saccharide according to the present invention is immobilized on said solid support by preferably covalent bonding.
  • the solid support is selected from the group comprising a glass slide, glass plate, a microtitre plate, microspheres, or beads.
  • the inventive saccharide is preferably covalently bound to the solid support using an interconnecting molecule.
  • the inventive saccharide can be used in immunological assays detection of for pneumonia, meningitis, otitis media, bacteremia and acute exacerbation of chronic bronchitis, sinusitis, arthritis and conjunctivitis caused by S. pneumoniae, preferably S. pneumoniae serotype 8.
  • Such assays comprise, for instance, microarray and ELISA useful for diagnosis of pneumonia, meningitis, otitis media, bacteremia and acute exacerbation of chronic bronchitis, sinusitis, arthritis and conjunctivitis caused by S. pneumoniae, preferably S. pneumoniae serotype 8.
  • Another aspect of the present invention refers to the use of a saccharide according to the present invention for diagnosis of pneumonia, meningitis, otitis media, bacteremia and acute exacerbation of chronic bronchitis, sinusitis, arthritis and conjunctivitis caused by S. pneumoniae, preferably S. pneumoniae serotype 8.
  • An assay conducted for diagnostic purposes according to the invention may be an immune assay like a solid-phase enzyme immunoassay (EIA), an enzyme linked immunosorbent assay (ELISA), especially an "indirect” ELISA or a radioimmune assay (RIA).
  • EIA solid-phase enzyme immunoassay
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmune assay
  • the saccharide according to the present invention is covalently linked on the solid support through an interconnecting molecule.
  • the saccharide according to the present invention can be covalently linked on the solid support directly or indirectly through the nitrogen atom of the -O-L-NH 2 group (see Figure 3 (C) ).
  • the solid support is preferably selected from the group comprising or consisting of: a glass slide, a microtitre plate, test tubes, microspheres, nanoparticle or beads.
  • the solid support is a glass slide or a microtitre plate.
  • a microtitre plate or microplate or microwell plate is a flat plate with multiple "wells" used as small test tubes. Typically, a microtitre plate having 6, 24, 96, 384 or even 1536 sample wells can be used.
  • Microplates are produced from many different materials, like polycarbonate for microtitre plate used for PCR. The most common is polystyrene as used for most optical detection microplates. It can be colored white by the addition of titanium dioxide for optical absorbance or luminescence detection or black by the addition of carbon for fluorescent biological assays.
  • Thioglycoside donor substrate 1 (6.0 g, 11.53 mmol) and acceptor with C-2 linker 2 (dried azeotropically using toluene in rotary evaporator, 3.93 g, 13.83 mmol) were taken in dry DCM (100 mL) and added 5 g of MW dried 4 ⁇ MS to it and stirred at rt for 15 min and then cooled to -10 °C.
  • NIS (3.83 g, 17.29 mmol) and TfOH (0.15 mL, 1.73 mmol) were then added to RM (reaction mixture) and stirred at -10 °C to -5 °C for 1 hr. Reaction completion was monitored by TLC.
  • RM was then quenched with 10% aq. Na 2 S 2 O 3 solution (50 mL) and then extracted with EtOAc (25 ml X 3). Combined organic layer was then washed with brine (10 ml), dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuum to get pale yellow oily compound. Crude product was purified on silica gel column chromatography using 20-30% EtOAc in hexanes to get spot which on evaporation yielded desired product 3 as pale yellow colored transparent gummy liquid (7.60 g, 89%).
  • Substrate 3 (7.50 g, 10.08 mmol) was taken in DCM (75 mL) under argon with activated 3 ⁇ MS for 10 min before cooling to 0 °C.
  • Extracted the aqueous with DCM (30 mL X 3) combined organics were washed thoroughly with water (20 mL X 3), brine (20 mL), dried over anhyd. Na 2 SO 4 , filtered, evaporated in vacuum to get colorless gummy solid.
  • Acceptor 4 (2.0 g, 2.68 mmol) was taken in DCM (30 mL) with activated 4 ⁇ AWMS and stirred at rt for 30 min before cooling to 0 °C. TMSOTf (0.49 ⁇ L, 0.27 mmol) was then added followed by the imidate donor 6 ( Carbohydrate Res 2008, 344, 439-447 .) (2.20 g, 3.89 mmol) in DCM (5 mL) over 5 min and the reaction mixture was stirred for 30 min at 0 °C. Quenched the RM with Et 3 N (1mL), filtered and the solvents removed under vacuum.
  • Substrate 5 (1.6 g, 1.317 mmol) was taken in pyridine (10 mL) at 0 °C and added HF-pyridine (3.56 mL, 39.5 mmol) to it and stirred at rt for 24 h. RM was washed with water and extracted with DCM (20 mL X 3). Combined organics were then washed with dil. HCl (50 mL X 2), sat.
  • reaction mixture was cooled (-20 °C) and TMSOTf (0.16 mL, 0.880 mmol, 0.115 eq.) was added. After stirring for 45 min, the reaction mixture was quenched by the addition of Et 3 N (1.0 mL). The organic solution was concentrated under vacuo.
  • TBS substrate 8 (2.0g, 2.018 mmol, 1 equiv.) was taken in pyridine (10 mL) at 0 °C and added 70% HF-pyridine (5.45 mL, 60.5 mmo, 30 equiv.) to it and stirred at rt for 36 h.
  • Substrate 13 (1.6g, 1.824 mmol, 1 equiv.) was taken in anhydrous DCM (10 mL) at 0°C and added pyridine (10 mL) and BzCl (0.635 mL, 5.47 mmol, 3 equiv.) to it dropwise and RM was stirred for 16 h.
  • RM was then evaporated in vacuum to remove solvents and then taken again in DCM (25 mL) and washed with aq.NaHCO 3 solution(5mL X 2). Organic layer was then dried on Na 2 SO 4 , filtered and evaporated in vacuum. which was then triturated using methanol to get off-white solid ( 12 ), filtered, dried in vacuum (1.5g, 84%).
  • Example 1-7 Synthesis of 2,3-di- O -benzoyl- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-2,3-di- O- benzoyl-6- O -benzyl- ⁇ -D-glucoyranosyl-(1 ⁇ 1)-(2- N -benzyl- N- benzyloxycarbonylamino)ethanol (14)
  • Example 1-8 Synthesis of methyl(2,3-di- O -benzoyl- ⁇ -D-glucopyranosyl)uronate-(1 ⁇ 4)-2,3-di- O -benzoyl-6- O -benzyl- ⁇ -D-glucoyranosyl-(1 ⁇ 1)-(2- N -benzyl- N -benzyloxycarbonylamino)ethanol (15)
  • Example 1-10 Synthesis of t Hexyl 4- O -benzoyl-2,3,6-tri- O -benzyl- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-2,3,6-tri- O -benzyl- ⁇ -D-galactopyranoside (44)
  • Example 1-11 Synthesis of 4- O -benzoyl-2,3,6-tri- O -benzyl- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-2,3,6-tri- O -benzyl- ⁇ -D-galactopyranosyl trifluoro-( N -phenyl)acetimidate (45)
  • Example 1-12 Synthesis of 4- O -benzoyl-2,3,6-tri- O -benzyl- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-2,3,6-tri- O -benzyl- ⁇ -D-galactopyranosyl-(1 ⁇ 4)-methyl[2,3-Di- O -benzoyl- ⁇ -D-glucopyranosyl]uronate-(1 ⁇ 4)-2,3-di- O -benzoyl-6- O -benzyl- ⁇ -D-glucoyranosyl-(1 ⁇ 1)-(2- N -benzyl- N -benzyloxycarbonylamino)ethanol (46)
  • Alcohol 15 100 mg, 87 ⁇ mol
  • imidate 45 121 mg, 105 ⁇ mol
  • the mixture was dissolved in Et 2 O (3.3 mL) and CH 2 Cl 2 (1.1 mL) and stirred over activated molecular sieves (3 ⁇ -AW) for 30 min at room temperature.
  • the solution was cooled to -20 °C and treated with TMSOTf (3.2 ⁇ L, 17 ⁇ mol). The mixture was stirred for 1 h and slowly warmed to 0 °C. The reaction was quenched with sat. aq.
  • Example 1-13 Synthesis of ⁇ -D-glucopyranosyl-(1 ⁇ 4)- ⁇ -D-galactopyranosyl-(1 ⁇ 4)- ⁇ -D-glucopyranosyluronic acid-(1 ⁇ 4)- ⁇ -D-glucoyranosyl-(1 ⁇ 1)-(2-amino)ethanol (10)
  • Example 1-14 Synthesis of 4- O -Benzoyl-2,3,6-tri- O -benzyl- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-2,3,6-tri- O -benzyl- ⁇ -D-galactopyranosyl-(1 ⁇ 1)-(2- N -benzyl- N- benzyloxycarbonylamino)ethanol (47)
  • Example 1-15 Synthesis of 2,3,6-tri-O-benzyl- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-2,3,6-tri-O-benzyl- ⁇ -D-galactopyranosyl-(1 ⁇ 1)-(2-N-benzyl-N-benzyloxycarbonylamino)ethanol (48)
  • Example 1-16 Syntheis of ⁇ -D-glucopyranosyl-(1 ⁇ 4)- ⁇ -D-galactopyranosyl-(1 ⁇ 1)-(2-amino)ethanol (49)
  • Example 1-17 Synthesis of 2,3-di- O -benzoyl- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-2,3-di- O -benzoyl-6- O -benzyl- ⁇ -D-glucoyranosyl-(1 ⁇ 4)-2,3,6-tri- O -benzyl- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-2,3,6-tri- O -benzyl- ⁇ -D-galactopyranosyl-(1 ⁇ 1)-(2- N- benzyl- N -benzyloxycarbonylamino)ethanol (50)
  • Alcohol 48 (47 mg, 41 ⁇ mol) and thioglycoside 12 (60 mg, 61 ⁇ mol) were co-evaporated with dry toluene (2x5 mL) and kept under high vacuum for 30 min.
  • the mixture was dissolved in CH 2 Cl 2 (2 mL) and stirred over activated molecular sieves (3 A-AW) for 30 min at room temperature.
  • the solution was cooled to -10 °C and treated with NIS (13.8 mg, 61 ⁇ mol) and triflic acid (1 ⁇ L, 11 ⁇ mol).
  • the mixture was kept for 1 h at that temperature and slowly warmed to 0 °C.
  • the reaction was quenched with Et 3 N (50 ⁇ L), filtered and concentrated to give the intermediate benzylidene acetal as a yellow oil.
  • Example 1-18 Synthesis of 2,3-di- O -benzoyl- ⁇ -D-glucopyranosyluronate-(1 ⁇ 4)-2,3-di- O -benzoyl-6- O -benzyl- ⁇ -D-glucoyranosyl-(1 ⁇ 4)-2,3,6-tri- O -benzyl- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-2,3,6-tri- O -benzyl- ⁇ -D-galactopyranosyl-(1 ⁇ 1)-(2- N- benzyl- N -benzyloxycarbonylamino)ethanol (51)
  • Example 1-19 Synthesis of ⁇ -D-Glucopyranosyluronic acid-(1 ⁇ 4)- ⁇ -D-glucoyranosyl-(1 ⁇ 4)- ⁇ -D-glucopyranosyl-(1 ⁇ 4)- ⁇ -D-galactopyranosyl-(1 ⁇ 1)- (2-amino)ethanol (18)
  • Example 1-20 Synthesis of methyl[2,3-di-O-benzoyl- ⁇ -D-glucopyranosyl]uronate-(1 ⁇ 4)-2,3-di-O-benzoyl-6-O-benzyl- ⁇ -D-glucoyranosyl-(1 ⁇ 4)-2,3,6-tri-O-benzyl- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-2,3,6-tri-O-benzyl- ⁇ -D-galactopyranosyl-(1 ⁇ 1)-(2-N-benzyl-N-benzyloxycarbonylamino)ethanol (52)
  • Example 1-21 Synthesis of 2,3,4,6-tetra- O -benzyl- ⁇ -D-galactopyranosyl-(1 ⁇ 4)-methyl[2,3-di- O -benzoyl- ⁇ -D-glucopyranosyl]uronate-(1 ⁇ 4)-2,3-di- O -benzoyl-6- O -benzyl- ⁇ -D-glucoyranosyl-(1 ⁇ 4)-2,3,6-tri- O -benzyl- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-2,3,6-tri- O -benzyl- ⁇ -D-galactopyranosyl-(1 ⁇ 1)-(2- N -benzyl- N- benzyloxycarbonylamino)ethanol (53)
  • Alcohol 52 14 mg, 7 ⁇ mol
  • thioglycoside 54 J Org Chem 1990, 55, 2860 .
  • 3x10 mL dry toluene
  • the mixture was dissolved in Et 2 O (1.05 mL) and CH 2 Cl 2 (0.35 mL) and stirred over activated molecular sieves (3 A-AW) for 30 min at room temperature.
  • the solution was cooled to -20 °C and treated with NIS (6.3 mg, 28 ⁇ mol) and TMSOTf (1 ⁇ L, 5.5 ⁇ mol). The mixture was stirred for 1 h and slowly warmed to 0 °C.
  • Example 1-22 Synthesis of ⁇ -D-galactopyranosyl-(1 ⁇ 4)- ⁇ -D-glucopyranosyluronate-(1 ⁇ 4)- ⁇ -D-glucoyranosyl- ⁇ -D-glucopyranosyl-(1 ⁇ 4)- ⁇ -D-galactopyranosyl-(1 ⁇ 1)-(2-amino)ethanol (55)
  • the intermediate carboxylic acid in CH 2 Cl 2 / t BuOH/water (1:16:8, 1 mL) was purged with argon and treated at 0 °C with a suspension of Pd(OH) 2 on carbon (20% (w/w) loading, 20 mg) in the same solvent mixture (0.5 mL).
  • the suspension was purged with hydrogen, stirred under hydrogen atmosphere for 16 h, filtered and concentrated. Since the reaction had not proceeded to completion, the residue was subjected to the same conditions again and stirred for 24 h at room temperature.
  • Example 1-23 Synthesis of 4- O -benzoyl-2,3,6-tri- O -benzyl- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-2,3,6-tri- O -benzyl- ⁇ -D-galactopyranosyl-(1 ⁇ 4)-methyl[2,3-di- O -benzoyl- ⁇ -D-glucopyranosyl]uronate-(1 ⁇ 4)-2,3-di- O -benzoyl-6- O -benzyl- ⁇ -D-glucoyranosyl-(1 ⁇ 4)-2,3,6-tri- O -benzyl- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-2,3,6-tri- O- benzyl- ⁇ -D-galactopyranosyl-(1 ⁇ 1)-(2- N -benzyl- N- benzyloxycarbonylamino)ethanol (56)
  • Alcohol 52 50 mg, 25 ⁇ mol
  • imidate 45 72.1 mg, 62 ⁇ mol
  • the mixture was dissolved in Et 2 O (2 mL) and CH 2 Cl 2 (0.67 mL) and stirred over activated molecular sieves (3 A-AW) for 30 min at room temperature.
  • the solution was cooled to -20 °C and treated with TMSOTf (2 ⁇ L, 11 ⁇ mol).
  • the mixture was stirred for 1 h and slowly warmed to 0 °C.
  • the reaction was quenched with sat. aq. NaHCO 3 (10 mL) and extracted with CH 2 Cl 2 (4x10 mL).
  • Example 1-24 Synthesis of ⁇ -D-glucopyranosyl-(1 ⁇ 4)- ⁇ -D-galactopyranosyl-(1 ⁇ 4)- ⁇ -D-glucopyranosyluronate-(1 ⁇ 4)- ⁇ -D-glucoyranosyl-(1 ⁇ 4)- ⁇ -D-glucopyranosyl-(1 ⁇ 4)- ⁇ -D-galactopyranosyl-(1 ⁇ 1)-(2-amino)ethanol (57)
  • the reaction was stirred for 20 h at that temperature, quenched with 10% aq. Na 2 SO 3 (0.8 mL) and concentrated under reduced pressure. The residue was dissolved in water (4 mL), neutralized with NaHSO 4 (0.5 M aq. solution) and extracted with EtOAc (4x10 mL). The combined organic fractions were dried over Na 2 SO 4 and concentrated. The residue was treated with NaOMe (0.5 M solution in MeOH, 1 mL), warmed to 40 °C and stirred for 5 h at that temperature. The reaction was cooled to room temperature, stirred for another 16 h at that temperature and treated with water (0.5 mL). The mixture was neutralized with Amberlite IR-120 (H + form), filtered and concentrated.
  • Example 1-25 Synthesis of ⁇ -D-Glucopyranosyl-(1 ⁇ 4)- ⁇ -D-glucoyranosyl-(1 ⁇ 4)- ⁇ -D-glucopyranosyl-(1 ⁇ 4)- ⁇ -D-galactopyranosyl-(1 ⁇ 1)-(2-amino)ethanol (60)
  • ester 50 (20 mg, 10.1 ⁇ mol) in THF (1 mL) and MeOH (0.33 mL) was added at room temperature NaOMe (0.5 M solution in MeOH, 0.5 mL). The reaction was warmed to 40 °C and stirred for 5 h at that temperature. The mixture was cooled to room temperature and stirred for 16 h at that temperature. The reaction was neutralized with Amberlite IR-120 (H + form), filtered and concentrated.
  • Example 1-26 Synthesis of 2,3-di- O -benzoyl- ⁇ -D-glucoyranosyl-(1 ⁇ 4)-2,3,6-tri- O -benzyl- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-2,3,6-tri- O -benzyl- ⁇ -D-galactopyranosyl-(1 ⁇ 1)-(2- N -benzyl- N -benzyloxycarbonylamino)ethanol (61)
  • Alcohol 48 (15 mg, 13 ⁇ mol) and thioglycoside 1 (20.4 mg, 39 ⁇ mol) were co-evaporated with dry toluene (2x5 mL) and kept under high vacuum for 10 min.
  • the mixture was dissolved in CH 2 Cl 2 (1.3 mL) and stirred over activated molecular sieves (3 A-AW) for 30 min at room temperature.
  • the solution was cooled to -20 °C and treated with NIS (8.8 mg, 39 ⁇ mol) and TfOH (1 ⁇ L, 11 ⁇ mol). The mixture was stirred for 1 h at that temperature and slowly warmed to 0 °C.
  • the reaction was quenched with a 1:1 (v/v) mixture of sat. aq.
  • Example 1-27 Synthesis of ⁇ -D-Glucoyranosyl-(1 ⁇ 4)- ⁇ -D-glucopyranosyl-(1 ⁇ 4)- ⁇ -D-galactopyranosyl-(1 ⁇ 1)-(2-amino)ethanol (62)
  • the combined organic phase was dried over MgSO 4 and the solvent was removed in vacuo.
  • the crude product was purified by silica gel flash column chromatography affording the title compound.
  • the crude was dissolved in DCM (6.5 mL) under an argon atmosphere.
  • 9-fluorenylmethyl chloroformate (8.3 g, 31.9 mmol) and pyridine (3.44 mL, 42.5 mmol) were added into the solution at 0 °C. After complete conversion of the starting material, the solution was diluted with DCM, extracted with 1 M aqueous HCl and saturated aqueous NaHCO 3 .
  • the combined organic phase was dried over MgSO 4 and the solvent was removed in vacuo.
  • Ethyl 2,3-di- O -benzyl-4,6- O -benzylidene-1-thio- ⁇ -D-galactopyranoside (38) was co-evaporated with toluene, and dissolved in DCM (6.5 mL) under an Ar atmosphere.
  • Triethylsilane (0.62 mL, 3.88 mmol) and trifluoroacetic anhydride (0.27 mL, 1.94 mmol) were added and the solution was cooled to 0 °C.
  • Trifluoroacetic acid (0.30 mL, 3.88 mmol) were added dropwise, and the reaction was stirred and allowed to warm to room temperature.
  • Methyl (2-methyl-5-tert-butyl-phenyl)-2-O-benzoyl-3-O-benzyl-4-O-fluorenylmethoxycarbonyl-1-thio- ⁇ -D-glucopyranosyluronate (42) was synthesized according to the procedure described in Angew. Chem. Int. Ed. 2013, 52, 5858 .
  • N-Iodosuccinimide (1.48 g, 6.66 mmol) and TfOH (60 ⁇ L, 0.66 mmol) was dissolved in a mixture of DCM (20 mL) and dioxane (20 mL).
  • Fmoc Deprotection Solution A solution of 20% triethylamine in DMF (v/v) was prepared.
  • Thioglycoside Building Block Solution 0.25 mmol of building block was dissolved in 2 ml of DCM.
  • the flow reactor setup consists of a medium pressure Hg lamp (Hanovia) with arc lengths of 27.9 cm and power of 450 W surrounded by a UV filter (Pyrex, 50% transmittance at 305 nm) in a quartz glass cooling system connected to a chiller to maintain a reaction temperature of 25 °C.
  • a fluorinated ethylene propylene (FEP) tubing (inner diameter: 0.03 inch; volume: 12 mL) is wrapped around the cooling system.
  • a syringe pump is connected to the FEP tubing and is used to flush solvents and resin via the inlet through the reactor.
  • the solid support is filtered off by a frit and the product solution is pooled and the solvents are removed in vacuo.
  • the UV lamp is located in a box that is additionally cooled by a fan.
  • the resin is washed with DCM (six times), swollen in 2 mL DCM and transferred into a disposable syringe (20 mL).
  • the FEP tubing is washed with 15 mL MeOH, then subsequently with 15 mL DCM using a flow rate of 4 mL ⁇ min-1.
  • the resin is slowly injected from the disposable syringe (20 mL) into the reactor and pushed through the tubing with 15 mL DCM (flow rate: 500 ⁇ L ⁇ min-1).
  • the tubing is washed with 20 mL DCM (flow rate: 500 ⁇ L ⁇ min-1).
  • the suspension leaving the reactor is directed into a filter where the resin is filtered off and washed with DCM.
  • the tubing is re-equilibrated with 15 mL DCM using a flow rate of 4 mL ⁇ min-1. The entire procedure is performed twice.
  • the resulting solution is evaporated in vacuo and the crude product is purified by HPLC (column: Luna silica; flow rate: 5 mL ⁇ min-1).
  • Example 1-33 The functionalized resin of Example 1-33 (65 mg; loading 0.385 mmol/g; 0.025 mmol) was loaded into the reaction vessel of the synthesizer and swollen in 2 mL DCM. To start the synthesis sequence, the resin was washed consecutively with DMF, THF, then DCM (three times each with 2 mL for 25 s). Module 1 for each building block and module 2 for Fmoc deprotection were performed to produce each saccharide structure.
  • the resin was cleaved from the solid support.
  • the crude product was purified by semi-preparative HPLC (column: Luna-Silica (21 ⁇ 250 mm; 5 ⁇ m); flow rate: 5 mL/min; eluents: Hexane/Ethyl acetate; gradient: 20% (5 min) ⁇ 60% (in 45 min) ⁇ 100% (in 5 min); detection: 210 and 280 nm) affording the target oligosaccharide.
  • the crude product was purified by semi-preparative HPLC (column: Luna-Hyper-Carbon (21 ⁇ 250 mm; 5 ⁇ m); flow rate: 5 mL/min; eluents: 0.1 % formic acid in Water / 0.1 % formic acid in acetonitrile; gradient: 10% (5 min) ⁇ 40% (in 30 min) ⁇ 100% (in 5 min); detection: ELSD) affording the target oligosaccharide.
  • Example 1-34 Synthesis of N-benzyloxycarbonyl-5-amino-pentanyl methyl 2-O-benzoyl-3- O -benzyl- ⁇ -D-glucopyranosyluronate-(1 ⁇ 4)-2- O -benzoyl-3,6-di- O- benzyl- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-6- O -acetyl-2,3-di- O -benzyl- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-2,3,6-tri- O -benzyl- ⁇ -D-galactopyranoside (19a)
  • Example 1-35 Preparation of 5-amino pentanyl ⁇ -D-glucopyranosyl uronic acid-(1 ⁇ 4)- ⁇ -D-glucopyranosyl-(1 ⁇ 4)- ⁇ -D-glucopyranosyl-(1 ⁇ 4)- ⁇ -D-galactopyranoside (19)
  • Tetrasaccharide 19a was subjected to the general deprotection procedure to afford tetrasaccharide 19: 36%; MS ESI+-HRMS m/z [M+H]+ calcd for C 29 H 52 NO 22 766.2975, found 766.2988.
  • Example 1-36 Synthesis of N-benzyloxycarbonyl-5-amino-pentanyl 4- O -acetyl-2,3,6-tri- O -benzyl- ⁇ -D-galactopyranosyl-(1 ⁇ 4)-methyl 2- O -benzoyl-3- O -benzyl- ⁇ -D-glucopyranosyluronate-(1 ⁇ 4)-2- O -benzoyl-3,6-di- O -benzyl- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-6- O -acetyl-2,3-di- O -benzyl- ⁇ -D-glucopyranoside (20a)
  • Example 1-37 Preparation of 5-amino pentanyl ⁇ -D-galactopyranosyl-(1 ⁇ 4)- ⁇ -D-glucopyranosyluronic acid-(1 ⁇ 4)- ⁇ -D-glucopyranosyl-(1 ⁇ 4)- ⁇ -D-glucopyranoside (20)
  • Tetrasaccharide 20a was subjected to the general deprotection procedure to afford tetrasaccharide 20: 40% MS ESI+-HRMS m/z [M+H]+ calcd for C 29 H 52 NO 22 766.2975, found 766.2964.
  • Example 1-38 preparation of N-benzyloxycarbonyl-5-amino-pentanyl 3,6-di- O- acetyl-2,4-di- O -benzyl- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-2,3,6-tri- O -benzyl- ⁇ -D-galactopyranosyl-(1 ⁇ 4)-methyl 2- O -benzoyl-3- O -benzyl- ⁇ -D-glucopyranosyluronate-(1 ⁇ 4)-2- O -benzoyl-3,6-di- O -benzyl- ⁇ -D-glucopyranoside (21a):
  • Example 1-39 preparation of 5-Amino pentanyl ⁇ -D-glucopyranosyl-(1 ⁇ 4)- ⁇ -D-galactopyranosyl-(1 ⁇ 4)- ⁇ -D-glucopyranosyluronic acid-(1 ⁇ 4)- ⁇ -D-glucopyranoside (21)
  • Tetrasaccharide 21a was subjected to the general deprotection procedure to afford tetrasaccharide 21: 42% MS ESI+-HRMS m/z [M+H]+ calcd for C 29 H 52 NO 22 766.2975, found 766.2977.
  • Example 1-40 Synthesis of N-benzyloxycarbonyl-5-amino-pentanyl 2-O-benzoyl-3,6-di- O -benzyl- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-6- O -acetyl-3,4-di- O -benzyl- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-2,3,6-tri- O -benzyl- ⁇ -D-galactopyranosyl-(1 ⁇ 4)-methyl 2- O -benzoyl-3- O -benzyl- ⁇ -D-glucopyranosyl uronate (22a)
  • Example 1-41 Synthesis of 5-amino pentanyl ⁇ -D-glucopyranosyl-(1 ⁇ 4)- ⁇ -D-glucopyranosyl-(1 ⁇ 4)- ⁇ -D-galactopyranosyl-(1 ⁇ 4)- ⁇ -D-glucopyranosyl uronic acid (22)
  • Tetrasaccharide 22a was subjected to the general deprotection procedure to afford tetrasaccharide 22: 52% MS ESI+-HRMS m/z [M+H]+ calcd for C 29 H 52 NO 22 766.2975, found 766.2988.
  • the indicated glycans were spotted onto CodeLink NHS slides using an automatic piezoelectric arraying robot (Scienion, Berlin, Germany) and incubated for 24 h (1% w/v in PBS) at room temperature. Slides were incubated in blocking buffer (100 mM ethanolamine in 50 mM NaPi pH > 9) for 30 min at room temperature, washed three times each with water and ethanol, and dried. Slides were then blocked with 1% (w/v) bovine serum albumin in phosphate buffered saline for 1 h at 37 °C, washed with water three times and dried.
  • blocking buffer 100 mM ethanolamine in 50 mM NaPi pH > 9
  • Example 2-2 Binding experiments using the microarrays synthesized according to the procedure described at example 2.1
  • Binding experiments were performed by incubating microarray slides coated with the saccharides of general formula ( I ) with either a rabbit anti-SP8 typing serum or human pneumococcal reference serum 007sp (pooled sera of 287 humans immunized with Pneumovax® vaccine purchase from National Institute for Biological Standards and Control) in the dilutions indicated in the presence or absence of native SP8 polysaccharide, and using fluorescently labeled anti-rabbit (goat anti-rabbit IgG-FITC, abcam ab6717) or anti-human secondary antibodies (Alexa Fluor 488 goat anti-human IgM, Invitrogen A21215; Alexa Fluor 647 goat anti-human IgG, Invitrogen A21445).
  • fluorescently labeled anti-rabbit goat anti-rabbit IgG-FITC, abcam ab6717
  • anti-human secondary antibodies Alexa Fluor 488 goat anti-human IgM, Invitrogen A21215; Alexa Fluor 647 goat
  • Example 2-3 Conjugation of synthetic tetrasaccharides 10 and 18 to CRM 197 using disuccinimidyl adipate:
  • the organic phase was discarded and the extraction step was repeated two times.
  • the aqueous layer was clarified by centrifugation in a 1.5 mL reaction tube (1 min, 14500 g, room temperature) and added to a stirring solution of CRM 197 (1 mg, 17.3 nmol) in NaPi (1 mL). The mixture was stirred for 16 h at room temperature and dialyzed using a centrifugal filter (10 kDa MWCO, Millipore, Darmstadt, Germany).
  • the conjugate was characterized by MALDI-MS:
  • mice (6-8 week old female Balb/c mice, Charles River) were immunized s. c. with CRM-Sp8 conjugates obtained at example 2.3 (corresponding to 4 ⁇ g synthetic glycan) formulated either with Freund's adjuvant (Sigma-Aldrich, St. Louis, US), Alum (Alhydrogel, Brenntag) or without adjuvant at a total volume of 100 ⁇ L at days 0, 14 and 28.
  • the immune response was monitored by glycan array.
  • the conjugate 59 induced an oligosaccharide-specific immune response in mouse #1160 receiving the said conjugate formulated with Freund's adjuvant.
  • a robust immune response was observed in immune serum from mouse #1160 against the native Sp8 polysaccharide (see Figure 5 ).
  • Monoclonal antibodies were prepared using BM-Condimed H1 (Roche, Penzberg, Germany) according to the manufacturer's instructions. Following fusion, single clones were generated using limited dilution and two subsequent rounds of subcloning. Antibody production was monitored by glycan array and ELISA. 33 clones were eventually isolated that produced mAbs recognizing both tetrasaccharide 19 and Sp8 native polysaccharide.
  • Clones 1H8 and 1F1 were expanded in serum-free medium.
  • MAb 1H8 was purified from the cell culture supernatant using a Protein G Antibody Purification kit (Pro-Chem, Littleton, USA) (see Figure 6 ).
  • MAb 1F1 was purified by gel filtration chromatography using a HiLoad 16/60 Superdex column (GE Healthcare, Little Chalfont, UK) with PBS as buffer.
  • Example 2-6 Enzyme-linked immunosorbent assay (ELISA):
  • ELISA was performed using high-binding polystyrene 96-well plates (Corning, Corning, US). Plates were coated using native Sp8 polysaccharide (SSI Diagnostica, Kopenhagen) at a concentration of 10 ⁇ g/mL in PBS for 20 h at 4 °C. Plates were blocked with 10% (v/v) fetal calve serum in PBS for 1 h at 37 °C and washed once with PBS containing 0.1% (v/v) Tween 20 (PBS-T). Cell culture supernatants of anti-Sp8 mAbs (50 ⁇ L) were applied.
  • native Sp8 polysaccharide SSI Diagnostica, Kopenhagen
  • HRP horseradish peroxidase
  • Polysaccharides were used at a concentration of 10 ⁇ g/mL and saccharide 18 at 20 ⁇ M in PBS.
  • Monoclonal antibody mAb 1H8 specifically recognizes both synthetic saccharide 18 and the native Sp8 polysaccharide (see Figure 7 ).
  • Example 2-8 Immunofluorescence of UV-inactivated S. pneumoniae:
  • S.pneumoniae serotype 8 (ATCC 6308) or serotype 1 (ATCC 6301) (approx. 4x10 8 cfu/mL) were inactivated by irradiation with 254 nm for 10 min in PBS at room temperature. Cells were harvested by centrifugation, washed once with PBS and frozen in Todd Hewitt Broth containing 0.5% (w/v) yeast extract and 20% (v/v) glycerol.
  • bacteria 8x10 8 cfu ST8 or 4x10 8 cfu ST1 were thawed, harvested by centrifugation (16800 g, 15 min, r.t.) and washed once in buffer A (50 mM NaHCO 3 , 100 mM NaCl, pH 7.5). Cells were resuspended in buffer A (1 mL) and treated with a fluorescein isothiocyanate (FITC, Sigma-Aldrich) solution (10 mg/mL in DMSO) to a final concentration of 0.1 mg/mL.
  • FITC fluorescein isothiocyanate
  • Bacteria were labeled in the dark for 1 h at 37 °C, harvested by centrifugation and washed twice with 0.25% (w/v) BSA in PBS (1 mL). Labeling was monitored by fluorescence microscopy using an Axio Imager.M2 system equipped with a LSM 700 confocal laser scanning microscope (Carl Zeiss Microscopy GmbH, Jena, Germany). Cells were suspended in 1% (w/v) BSA in PBS (1 mL for ST8, 0.5 mL for ST1) and the suspension was distributed into two aliquots.
  • the suspensions were treated with mAb 1H8 or mAb 1E12 (IgG1) against Yersinia pestis lipopolysaccharide core trisaccharide as an isotype control to a final mAb concentration of 10 ⁇ g/mL.
  • Bacteria were incubated in the dark for 16 h at 4 °C under agitation and washed with 1% (w/v) BSA in PBS (0.5 mL).
  • the cells were suspended in a solution of goat anti-mouse IgG-Alexa635 conjugate (1:100 dilution in 200 ⁇ L 1% (w/v) BSA in PBS, Invitrogen), incubated in the dark for 1.5 h at room temperature and washed with 1% (w/v) BSA in PBS and PBS (0.5 mL, respectively). Fluorescently labeled bacteria were visualized by fluorescence microscopy and images were processed with using Zen 2011 software (Carl Zeiss Microscopy GmbH). As shown in Figure 8 , immunization with conjugate 59 induces the formation of antibodies that recognize the native SP-8 polysaccharide and ST8 bacteria.
  • Example 2-9 Assessment of the binding of mAbs 1H8 and 1F1 to S . pneumoniae serotype 8 bacteria by flow cytometry
  • S. pneumoniae serotype 8 (ATCC 6308), serotype 1 (ATCC 6301) or serotype 3 (PN36, NCTC7978) were UV-inactivated, FITC-labeled and treated with a fluorescent secondary antibody (anti-mouse IgG-Alexa635 conjugate or anti-mouse IgM-Alexa680 conjugate, Invitrogen) as described before (see Example 2.8 ).
  • Flow cytometry was performed using a FACSCanto II flow cytometer (BD Pharmingen, Heidelberg, Germany) and analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA). Both monoclonal antibodies 1H8 and 1F1 bind to S .
  • opsonophagocytotic killing assay was employed as described in Romero-Steiner et al., Clin. Diagn. Lab. Immunol., 1997, 4. Briefly, HL-60 cells were differentiated for one week with N , N -dimethylformamide as reported (Romero-Steiner et al., 1997), washed twice with OPKA buffer (Hanks' buffer with 0.1% (w/v) gelatin) and diluted to a density of 10 7 cells/mL in the same buffer directly before use.
  • OPKA buffer Hors' buffer with 0.1% (w/v) gelatin
  • Bacteria were grown in growth medium (Todd-Hewitt broth + 0.5% (w/v) yeast extract) at 37 °C/5% CO 2 to log phase (OD 0.2-0.3), diluted in freezing medium (growth medium with 15% (v/v) glycerol) to a density of approx. 10 6 cells/mL and frozen in 0.5 mL aliquots at -80 °C.
  • Bacteria were diluted with OPKA buffer and aliquoted (1000 cells in 20 ⁇ L each) in a 96 well-plate. Bacterial suspensions were treated with appropriate antibody or antisera (1:4) dilutions and incubated for 15 min at 37 °C.
  • Complement source baby rabbit complement, CedarLane, 10 ⁇ L
  • differentiated HL-60 cell suspension 40 ⁇ L, phagocyte/bacteria ratio 400:1
  • Opsonophagocytosis was performed in triplicates. 10% of the contents of each well were plated on Columbia Agar plates and colonies were counted after 10-12 h incubation at 37 °C/5% CO 2 . Control wells lacked either antibody or complement sources.
  • Example 2-11 Glycan array analysis using the monoclonal antibodies mAbs 1H8 and 1F1.
  • Binding experiments were performed by incubating microarray slides synthesized as shown in Example 2.1 in the presence or absence of SP8 capsular polysaccharide and including compounds 19, 49, 90, 60, 62, 55 and 57.
  • Example 2-12 Assessment of the binding of mAb 28H11 to the saccharides of the present invention by microarray
  • Glycan arrays were fabricated as shown above ( Example 2-1 and 2-2 ), except that a murine monoclonal antibody 28H11 (IgM) raised against native Sp8 polysaccharide ( Yano and Pirofski (2011), Clin. Vaccine Immunol., 18 (1), 59-66 ) was used for binding at different dilutions with or without addition of 10 ⁇ g/mL S. pneumoniae type 8 CPS.
  • a Donkey anti-Mouse IgM Alexa Fluor® 594 conjugate (dianova, Hamburg, Germany) was used as a secondary antibody for detection.
  • MAb 28H11 a murine IgM that has been raised against native S . pneumoniae type 8 CPSs, is well-characterized and protects mice from infection with live S . pneumoniae type 8 pneumococci in various settings ( Yano and Pirofski (2011), Clin. Vaccine Immunol., 18 (1), 59-66 ).
  • Glycan microarray analysis revealed a robust interaction of mAb 28H11 with saccharide 19 that was specific to S. pneumoniae type 8, as shown by the ablation of binding by native S. pneumoniae type 8 CPSs of up to 10 ⁇ g/mL (see Figure 12 ).
  • Example 2-13 Conjugation of a oligosaccharides to CRM 197 using bis(4-nitrophenyl)adipate:
  • CRM 197 (3 mg, 52 nmol) was dialyzed twice against 0.1 M sodium phosphate buffer pH 8.0 using a centrifugal filter (10 kDa MWCO, Millipore, Darmstadt, Germany), concentrated to approx. 300 ⁇ L and added to the activated oligosaccharide. The mixture was stirred at room temperature for 16 h and dialyzed (see above) four times against water. An aliquot was taken for characterization and the mixture was dialyzed three times against phosphate-buffered saline. The glycoconjugates were characterized by MALDI-TOF MS (see Figure 13 ), SDS-PAGE and Western Blot.
  • Tetrasaccharide 18 2.6 ⁇ mol (1.9 mg)
  • Tetrasaccharide 60 2.1 ⁇ mol (1.5 mg)
  • Hexasaccharide 57 1.9 ⁇ mol (2.0 mg)
  • Conjugate CRM 197 -18 ca. 65503 m / z (incorporation of 8.8 tetrasaccharide molecules on average); Conjugate CRM 197 -60: ca. 68281 m / z (incorporation of 12.9 tetrasaccharide molecules on average); Conjugate CRM 197 -57: ca. 63535 m / z (incorporation of 4.6 hexasaccharide molecules on average).
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CN108367063A (zh) * 2015-07-21 2018-08-03 辉瑞公司 包含缀合的荚膜糖抗原的免疫原性组合物及其试剂盒和用途
US10864261B2 (en) * 2015-12-17 2020-12-15 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Synthetic vaccines against Streptococcus pneumoniae serotype 2
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